Microfluidic determination of low abundance events

ABSTRACT

Provided are microfluidic systems and methods for detecting, sorting, and dispensing of low abundance events such as single cells and particles, including a variety of eukaryotic and bacterial cells, for a variety of bioassay applications. The systems and methods described herein, when implemented in whole or in part, will make relevant microfluidic based tools available for a variety of applications in biotechnology including antibody discovery, immuno-therapeutic discovery, high-throughput single cell analysis, target-specific compound screening, and synthetic biology screening.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 17/089,052, filed on Nov. 4, 2020, which application is acontinuation of International Application No. PCT/US2020/035340, filedMay 29, 2020, which claims the benefit of U.S. Provisional ApplicationNo. 62/855,734, filed May 31, 2019, the entire contents of which areincorporated herein by reference.

BACKGROUND

Single cell analysis technologies are critical to biotechnologicalresearch and development due to the complex heterogeneity of cells, andtheir interconnectedness with each other. A widely used single cellanalysis tool is flow cytometry (FC), which is able to analyzeindividual cells according to their size, shape, and the fluorescenceproperties of cell surface and intracellular markers. It is calledfluorescence activated cell sorting (FACS) when the device also enablesthe sorting of specific cells from a heterogeneous cell population.

The great success of FACS is in part due to its high throughput forscreening individual single cells based on fluorescent detection at upto tens of thousands of cells per second. However, FACS can neither beused to probe secreted factors from individual single cells, nor probethe interactions between two single cells.

A variety of microfluidic technologies have been developed for singlecell analysis, including microchambers, micro-wells, and droplets.Microchambers and nano-wells have limited applications due to theirrelatively low throughput. In the past decade, droplet microfluidics hasgained increasingly more attention. Droplet microfluidics are uniquelyadvantageous due to the ultra-small assay volume (usually less than 1nanoliter (nL)), flexible throughput (thousands to hundreds of millionsof cells), and maneuverability such as merging, splitting, trapping,detecting, and sorting, which fit well for many biological assays ofindividual single cells including genomic analysis and live cell assays.

Despite the progress of droplet technologies, there remain major bottlenecks that limit important applications requiring highly accurate andefficient single cell or particle detection and isolation. For example,antigen-specific high-quality T or B cells are generally low abundanceevents among a T or B cell immune repertoire, respectively. There aresignificant unmet demands to further improve the accuracy and efficiencyof current droplet technologies, which will enable the effectiveisolation of such low abundance events prevalent in many biologicalapplications.

SUMMARY

It would therefore be desirable to provide devices, systems, and methodswhich enable more accurate and efficient detecting, sorting, anddispensing of low abundance events such as single cells and particles.Not necessarily all such aspects or advantages are achieved by anyparticular embodiment. Thus, various embodiments may be carried out in amanner that achieves or optimizes one advantage or group of advantagestaught herein without necessarily achieving other aspects or advantagesas may also be taught or suggested herein.

The present disclosure is related to systems and methods for detecting,sorting, and dispensing droplets in bioassays, including determining lowabundance events such as rare single-cell clones and entities that arepresent in a complex biological sample.

The following summary is illustrative only and is not intended to belimiting in any way. That is, the following summary is provided tointroduce concepts, highlights, benefits and advantages of the novel andnon-obvious techniques described herein. Select implementations arefurther described below in the detailed description. Thus, the followingsummary is not intended to identify essential features of the claimedsubject matter, nor is it intended for use in determining the scope ofthe claimed subject matter.

Provided are methods, modules and systems for detecting, sorting, anddispensing water-in-oil droplets or emulsions comprising cell(s) and/orparticle(s) in a microfluidic system. Provided are advanced modules,systems and methods for highly efficient sorting and dispensing ofsingle cells in low abundance events related applications using one, twoor more detection points and/or serial sorting. Provided are also themethods and systems for synchronizing droplet detection and dispensingin support of the post-processing downstream analyses.

In a first aspect, a system for detecting, sorting, and dispensingdroplets for use in bioassays is provided. The system comprises amicrofluidic device comprising a first channel connected to a secondchannel and a waste channel by a first sorting junction; a plurality ofwater-in-oil droplets, wherein at least two of the plurality of dropletseach comprise at least one cell, at least one particle, or at least onecell plus at least one particle; a first detector or sensorcorresponding to a first point of detection disposed along the firstchannel upstream of the sorting junction, wherein the first detectorcomprises an optical detector; a second detector or sensor correspondingto a second point of detection disposed along the second channeldownstream of the sorting junction; a target droplet dispensing modulecomprising a dispensing nozzle disposed downstream of the second pointof detection; and a processor configured to index each of a plurality oftarget droplets dispensed by the dispensing nozzle with an opticalsignal of the same target droplet detected by a) the first detector orsensor at the first point of detection, b) the second detector or sensorat the second point of detection, or c) both the first detector orsensor and the second detector or sensor.

In some embodiments, the system may further comprise a dropletgeneration module, a droplet incubation module, or a droplet generationplus a droplet incubation module.

In some embodiments, the at least one cell may be a mammalian cell, aeukaryotic cell, a yeast cell, a bacterial cell, a primary cell, animmortalized cell, a cancer cell, a hybrid cell, or a derivative or anengineered form thereof.

In some embodiments, the at least one particle may be a microparticle ora nanoparticle.

In some embodiments, the optical detector may comprise a photomultiplier tube (PMT), a camera-like device, a charge coupled device(CCD) camera, a complementary metal-oxide semiconductor (CMOS) camera,or an avalanche photodiode detector (APD).

In some embodiments, the second detector or sensor may comprise anoptical sensor or a non-optical sensor. The optical sensor ornon-optical sensor may be configured to detect the presence of a dropletin a non-discriminative manner with respect to the at least one cell orat least one particle.

In some embodiments, the second detector or sensor may comprise anoptical sensor or a non-optical sensor. The optical sensor ornon-optical sensor may be configured to detect the presence of adroplet, a relative speed of the droplet, and/or a size of the dropletin the second channel.

In some embodiments, the second detector or sensor may comprise anoptical detector or a non-optical detector.

In some embodiments, the second detector or second sensor may comprisean optical detector. In some embodiments, the second detector or secondsensor may comprise a photo multiplier tube (PMT), a camera, acamera-like device, a camera-like detector, a charge coupled device(CCD) camera, a complementary metal-oxide semiconductor (CMOS) camera,or an avalanche photodiode detector (APD).

In some embodiments, the second detector or sensor may be configured todetect two or more optical signals (e.g., images) for each of aplurality of target droplets, wherein the two or more optical signals(e.g., images) detected by the second detector or sensor comprise thesecond signal from the second point of detection. In some embodiments,the two or more images for each of the plurality of target droplets maycomprise a signal generated by a modulated or pulsed light sourceconfigured to provide repetitive short illumination of light energy. Insome embodiments, the modulated or pulsed light source may optionallycomprise one or more lasers or laser-like sources configured to providestroboscopic illumination.

In some embodiments, the system may comprise an optical assemblyconfigured to provide a short illumination for generating one of the twomore optical signals at the second point of detection. The duration ofthe short illumination may be within a range of about 0.5 to about 50milliseconds, or about 5 to about 30 milliseconds. In some embodiments,the optical assembly may comprise a modulated or pulsed laser source,and wherein the short illumination comprises stroboscopic illuminationprovided by the modulated or pulsed laser source. In some embodiments,the first detector or sensor may be configured to provide a precisetiming trigger to the optical assembly to trigger the stroboscopicillumination. Alternatively, or in combination, the processor may beconfigured to synchronize the first detector or sensor with triggeringof modulated or pulsed light source to repetitively illuminate (such aswith stroboscopic illumination) the second point of detection.

In some embodiments, the system may comprise an optical assemblyconfigured to provide droplet imaging (e.g., with stroboscopicillumination) at a point of detection (e.g., the first or the secondpoint of detection). An upstream detector or sensor (e.g., the firstdetector or sensor or a third detector or sensor) may be configured todetect or sense a droplet upstream of the point of detection in order toprovide a first signal to trigger illumination (such as stroboscopicillumination) at an appropriate timing to image a droplet with highspatiotemporal resolution at the designated point of detection (e.g., togenerate a second signal). Such a signal generated by imaging can beused to inform subsequent droplet sorting and/or droplet dispensing. Aprocessor may be configured to synchronize the sorting and/or dispensingmechanism with one or more of the first and the second detectors orsensors based on one or more of the first and the second signals/images.

In some embodiments, the second detector or second sensor may comprise anon-optical detector configured to detect non-optical signals. Thenon-optical signals may represent individual droplets. The non-opticalsignals may comprise contact conductivity, contactless conductivity,impedance, or magnetic force.

In some embodiments, the system may comprise one or more bypass channelsconnected to a main fluidic channel downstream of a sorting junction butupstream of a dispensing nozzle (i.e., this segment of fluidic channelis a “sorting channel”) The bypass channel may be further connected to awidened channel, compartment, or chamber (generally, a “buffer zone”)that may serve to reduce the speed of traveling droplets in the sortingchannel. In some embodiments, a serial or an array of pillars may beprovided at the interface between the bypass channels and the sortingchannel to constrain the droplets moving along the sorting channel.

In some embodiments, the at least one cell may be labelled with afluorophore or expresses a fluorescent molecule. Alternatively, or incombination, the at least one cell may express a luminescent orluminogenic molecule.

In some embodiments, the at least one particle may be labelled with afluorophore.

.In some embodiments, the second point of detection may be disposedabout 1 cm to about 60 cm upstream of the dispensing nozzle.

In some embodiments, any of the systems described herein may furthercomprise a third detector or sensor corresponding to a third point ofdetection disposed downstream of the second point of detection andupstream of the target droplet dispensing module. In some embodiments,the third point of detection may be disposed about 1 cm to about 60 cmupstream of the dispensing nozzle

In some embodiments, any of the systems described herein may comprise athird channel connected to the second channel and a second waste channelby a second sorting junction, the second sorting junction disposeddownstream of the first sorting junction and upstream of the targetdroplet dispensing module. The system may further comprise a thirddetector or sensor corresponding to a third point of detection disposeddownstream of the second sorting junction and upstream of the targetdroplet dispensing module. In some embodiments, the third point ofdetection may be disposed about 1 cm to about 60 cm upstream of thedispensing nozzle.

In some embodiments, any of the systems described herein may comprise alaser or a laser-like source. The laser or laser-like source can beconfigured to illuminate the first, second, and/or third point ofdetection. The laser-like source can comprise a light emitting diode(LED). In some embodiments, the system may further comprise a lasermodulator comprising a beam splitter comprising an optical elementconfigured to split an energy beam generated by the laser or laser-likesource into a first beam and a second beam. The optical element maydirect the first and second beams to the first or second point ofdetection to provide dual focusing at the first or second point ofdetection along the fluidic flow direction. In some embodiments, theoptical element of the beam splitter may comprise a fiber opticalsplitter that can split light into two outgoing laser beams. In someembodiments, the optical element of the beam splitter may comprise abirefringent polarizer such as a Wollaston prism, which can split lightinto two linearly polarized outgoing laser beams with orthogonal or nearorthogonal polarization.

In some embodiments, the system may further comprise an optical elementconfigured to provide dual focusing along the first channel at the firstpoint of detection. The optical element may comprise an optical fibersplitter or a birefringent polarizer configured to split an energy beamgenerated by one or more lasers or laser-like sources into a first beamand a second beam and direct the first and second beams to the firstpoint of detection.

In some embodiments, any of the systems described herein may comprise alaser modulator comprising a remote focusing device. The remote focusingdevice may comprise an optical element configured for remote focusingsuch that multiple focal planes at different axial positions along amicrofluidic channel (e.g., a first channel, a second channel, a thirdchannel, etc.) can be detected in rapid sequence or in parallel. Theoptical element of the remote focusing device may comprise an electricallens or a tunable acoustic gradient (TAG) index lens. Alternatively, orin combination, the system may further comprise a laser modulatorcomprising an optical element configured to generate a uniform,non-diffracting beam across the first or second channel at the first orsecond point of detection, respectively. The optical element maycomprise an axicon, an annular aperture, or a spatial light modulator.

In some embodiments, the target droplet dispensing module may beconfigured to dispense the target droplets into one or more collectiontubes or plates in a controlled manner. The one or more collection tubesor plates may comprise a 96-well plate, a 384-well plate, or amulti-well plate. In some embodiments, the dispensing module maycomprise an x-y-z moving dispenser, a rotatory dispenser, or thecombination thereof.

In some embodiments, the first signal or the second signal may comprisean optical signal, an electrical signal, or an optical signal plus anelectrical signal. The first signal or the second signal may beconfigured to synchronize one or more of the first point of detectionand/or the second point of detection with the dispensing nozzle.

In some embodiments, any of the systems described herein may furthercomprise a magnet, a pair of magnets, or an array of magnets adjacentthe first channel or the second channel. The magnet, pair of magnets, orarray of magnets may be positioned at a distance of about 0.01 mm toabout 30 mm from the first point of detection or the second point ofdetection. For example, the magnet, pair of magnets, or array of magnetsmay be positioned at a distance of about 0.1 mm to about 10 mm away fromthe first point of detection or the second point of detection. In someembodiments, the magnet, pair of magnets, or array of magnets maycomprise a permanent magnet, a tunable electric magnet, or thecombination thereof.

In some embodiments, the processor may be configured to synchronize thedispensing nozzle with one or more of the first or second detector orsensor based on one or more of the first signal or the second signal.

In another aspect, a system for detecting droplets in bioassays isprovided. The system comprises a microfluidic device comprising a firstchannel having a size of at least 35 μm, for example at least about 60μm, in each cross-sectional inner dimension; a plurality of water-in-oildroplets, at least two of the plurality of droplets comprising at leastone cell, at least one particle, or at least one cell plus at least oneparticle; a prism adjacent to the first channel; a first objectivedisposed at a first corner of the prism; a second objective disposed ata second corner of the prism, the second objective disposed at an anglerelative to the first objective; a laser configured to generate a laserenergy; and a laser modulator comprising a remote focusing unit or anoptical element generating a non-diffracting beam, wherein the lasermodulator is configured to modulate the laser energy before it entersone or more of the first objective or the second objective. At least oneof the first objective or second objective is configured to direct themodulated laser energy to illuminate the plurality of droplets that passthrough a first detection point of the first channel.

In some embodiments, the system may further comprise a droplet sortingmodule. Alternatively, or in combination, the system may furthercomprise a dispensing module.

In some embodiments, the angle may be about 60 degrees to about 120degrees. For example, the angle may be about 80 degrees to about 100degrees. Preferably, the angle may be about 90 degrees.

In some embodiments, the first objective or the second objective may beconfigured to direct the modulated laser energy at an angle of about 30degrees to about 60 degrees relative to the first channel. For example,the first objective or the second objective may be configured to directthe modulated laser energy at an angle of about 40 degrees to about 50degrees. Preferably, the first objective or the second objective may beconfigured to direct the modulated laser energy at an angle of about 45degrees.

In some embodiments, the remote focusing unit may comprise a tunableacoustic gradient (TAG) index lens.

In some embodiments, the optical element generating a non-diffractingbeam may comprise an axicon, an annular aperture, or a spatial lightmodulator.

In some embodiments, the prism may comprise a material having arefractive index of about 1.28 to about 1.6. For example, the prism maycomprise a material having a refractive index of about 1.29 to about1.58.

In another aspect, a system for detecting, sorting, and dispensingdroplets is provided. The system comprises a microfluidic devicecomprising a first channel connected to a second channel and a wastechannel by a first sorting junction; a plurality of water-in-oildroplets, at least two of the plurality of droplets each comprise atleast one cell, at least one particle, or at least one cell plus atleast one particle; a first detector or sensor corresponding to a firstpoint of detection disposed along the first channel upstream of thefirst sorting junction; a second detector or sensor corresponding to asecond point of detection disposed along the second channel downstreamof the first sorting junction, wherein the second detector or sensor isconfigured to detect two or more images for each of a plurality oftarget droplets; a sorting module; and a droplet dispensing modulecomprising a dispensing nozzle disposed downstream of the second pointof detection.

In some embodiments, the plurality of target droplets may be a firstbatch of target droplets and further sorting downstream or upstream ofthe second detector or sensor may generate a second batch of targetdroplets.

In some embodiments, the system may further comprise a processorconfigured to index each of the plurality of target droplets dispensedby the dispensing nozzle with the signal of the same target dropletdetected by the second detector or sensor at the second point ofdetection.

In some embodiments, the system may further comprise one or more lasersor laser-like light sources to generate illumination at the first pointof detection.

In some embodiments, the system may further comprise an optical elementconfigured to provide dual focusing along the first fluidic channel atthe first point of detection. The optical element may comprise anoptical fiber splitter or a birefringent polarizer. The optical elementmay be configured to split an energy beam generated by the one or morelasers or laser-like sources into a first beam and a second beam anddirect the first and second beams to the first point of detection.

In some embodiments, the first detector or sensor may comprise afast-response optical detector. The fast-response optical detector maycomprise a photo multiplier tube (PMT), a photodiode, or an avalanchephotodiode detector (APD).

In some embodiments, the second detector or sensor may comprise a cameraor a camera-like detector.

In some embodiments, the two or more images for each of the plurality oftarget droplets may comprise a signal generated by a modulated or pulsedlight source configured to provide repetitive short illumination oflight energy. In some embodiments, each duration of the repetitive shortillumination of light energy may last about 0.5 to about 50milliseconds, or about 5 to about 30 milliseconds. In some embodiments,the modulated or pulsed light source may optionally comprise one or morelasers configured to provide stroboscopic illumination. In someembodiments, the signal generated by stroboscopic illumination maycomprise a first signal. The first detector or sensor may be configuredto detect or sense a second signal from the first point of detection.The processor may be configured to synchronize the dispensing nozzlewith one or more of first or second detector or sensor based on one ormore of the first signal and the second signal. Alternatively, or incombination, the processor may be configured to synchronize the firstdetector or sensor with triggering of modulated or pulsed light sourceto repetitively illuminate (such as with stroboscopic illumination) thesecond point of detection.

In some embodiments, the system may further comprise an optical assemblyconfigured to provide repetitive short single-pulse or burst of pulsesof light energy such as stroboscopic illumination at the second point ofdetection. In some embodiments, the system may further comprise anupstream detector or sensor corresponding to a third point of detectiondisposed along the second channel between the first sorting junction andthe second point of detection. The upstream detector or sensor may beconfigured to provide a precise timing trigger to the optical assemblyto trigger the stroboscopic illumination. Alternatively, or incombination, the first detector or sensor may be configured to provide aprecise timing trigger to the optical assembly to trigger thestroboscopic illumination.

In some embodiments, the first detector or sensor may be configured todetect or sense a first signal from the first point of detection. Thetwo or more images detected by the second detector or sensor maycomprise a second signal from the second point of detection. A processormay be configured to synchronize the dispensing nozzle with one or moreof the first or second detector or sensor based on one or more of thefirst signal and the second signal.

In some embodiments, the system may comprise an optical assemblyconfigured to provide droplet imaging (e.g., with stroboscopicillumination) at a point of detection (e.g., the first or the secondpoint of detection). An upstream detector or sensor may be configured todetect or sense a droplet upstream of the point of detection in order toprovide a first signal to trigger illumination (such as stroboscopicillumination) at an appropriate timing to image a droplet with highspatiotemporal resolution at the designated point of detection (e.g., togenerate a second signal). Such a signal generated by imaging can beused to inform subsequent droplet sorting and/or droplet dispensing. Aprocessor may be configured to synchronize the sorting and/or dispensingmechanism with one or more of the first and the second detectors orsensors based on one or more of the first and the second signals/images.

Another aspect provides a system for droplet detecting, sorting, anddispensing. The system comprises a microfluidic device comprising (i) afirst channel connected to a second channel and a first waste channel bya first sorting junction and (ii) a third channel connected to thesecond channel and a second waste channel by a second sorting junctiondisposed downstream of the first sorting junction; a plurality ofwater-in-oil droplets, wherein at least two of the plurality of dropletseach comprise at least one cell, at least one particle, or at least onecell plus at least one particle; a multi-zone detection modulecomprising one or more detectors corresponding to a first point ofoptical detection disposed along the first channel and a second point ofoptical detection disposed along the second channel; a dropletdispensing module; and a processor configured to index each of aplurality of target droplets dispensed by the droplet dispensing modulewith an optical signal of the same target droplet detected by the firstoptical detector at the first point of detection or the second opticaldetector at the second point of detection.

In some embodiments, the one or more detectors may comprise a multi-zoneoptical detector with a single detection area in the microfluidic devicecomprising the first point of optical detection and the second point ofoptical detection. In some embodiments, at least a portion of the firstchannel or second channel disposed between the first point of opticaldetection and the second point of optical detection may comprise alooping channel that loops from the first point of optical detection tothe second point of optical detection. Alternatively, or in combination,the multi-zone optical detector may comprise a multi-channel photomultiplier or a camera.

In another aspect, a method for detecting, sorting and dispensingdroplets is provided. The method comprises providing a plurality ofwater-in-oil droplets to a first channel of a microfluidic device,wherein at least two of the plurality of droplets each comprise at leastone cell, at least one particle, or at least one cell plus at least oneparticle; flowing the plurality of droplets past a first point ofoptical detection disposed along the first channel; detecting a firstsignal from each of the plurality of droplets at the first point ofoptical detection; identifying a first batch of target droplets based onthe first signal; sorting the first batch of target droplets through asorting actuator into a second channel of the microfluidic device toobtain sorted droplets; flowing sorted droplets past a second point ofdetection or a sensor disposed along the second channel; detecting asecond signal from each of the sorted droplets at the second point ofdetection or sensor; identifying a second batch of target droplets basedon the second signal; dispensing the second batch of target dropletsindividually; and indexing the second batch of target droplets with oneor both of the first signal and the second signal such that each indexeddispensed droplet matches precisely one or both of the first signaldetected from each of the first batch of target droplets and the secondsignal detected from each of the sorted droplets.

In some embodiments, the method may further comprise generating theplurality of water-in-oil droplets, incubating the plurality ofwater-in-oil droplets, or generating the plurality of water-in-oildroplets plus incubating the plurality of water-in-oil droplets.

In some embodiments, detecting the first signal may comprise detectingan optical signal from the at least one cell, the at least one particle,or the at least one cell plus the at least one particle.

In some embodiments, detecting the second signal may comprise detectingan optical signal from the at least one cell, the at least one particle,or the at least one cell plus the at least one particle.

In some embodiments, detecting the second signal may comprise detectingan optical signal or a non-optical signal indicative of a presence ofone of the plurality of droplets within the second channel at the secondpoint of detection or sensor.

In some embodiments, the second point of detection or the sensor may bedisposed a distance of about 1 cm to about 60 cm upstream of adispensing nozzle of a dispensing module.

In some embodiments, the first signal may be generated based on dualfocusing along the first channel at the first point of detection.

In some embodiments, the method may further comprise illuminating thefirst point of optical detection and/or the second point of opticaldetection with one or more lasers or laser-like sources.

In some embodiments, the method may further comprise modulating a laserat the first point of detection by an optical element that provides dualfocusing. The dual focusing optical element may comprise an opticalfiber splitter or a birefringent polarizer.

In some embodiments, the method may further comprise modulating a laserat the first point of detection by a remote focusing device. The remotefocusing device may comprise an electrical lens or a tunable acousticgradient (TAG) index lens.

In some embodiments, the method may further comprise modulating a laserto generate a non-diffracting beam with an optical element. The opticalelement may comprise an axicon, an annular aperture, or a spatial lightmodulator.

In some embodiments, the second signal may comprise an optical signal ora non-optical signal.

In some embodiments, detecting the second signal may comprise detectingtwo or more images for each of the first batch of target droplets.

In some embodiments, dispensing may comprise dispensing the second batchof target droplets by a dispensing module into collection tubes orplates in a controlled manner. The plates may comprise a 96-well plate,a 384-well plate, or a multi-well plate.

In another aspect, a method for detecting, sorting and dispensingdroplets is provided. The method comprises providing a plurality ofwater-in-oil droplets to a first channel of a microfluidic device,wherein at least two of the plurality of droplets each comprise at leastone cell, at least one particle, or at least one cell plus at least oneparticle; flowing the plurality of droplets past a first point ofoptical detection disposed along the first channel; detecting a firstsignal from each of the plurality of droplets at the first point ofdetection, wherein the first signal is generated based on dual focusingalong the first channel at the first point of detection; identifying afirst batch of target droplets based on the first signal; sorting thefirst batch of target droplets into a second channel of the microfluidicdevice; flowing the first batch of target droplets past a second pointof detection disposed along the second channel; detecting a secondsignal from each of the first batch of target droplets at the secondpoint of detection, wherein the second signal is generated by imaging;identifying a second batch of target droplets, the second point ofdetection being based on spatial resolution such as imaging; anddispensing the second batch of target droplets.

In some embodiments, the method may further comprise indexing the secondbatch of target droplets such that each dispensed droplet matchesprecisely the second signal detected from each of the second batch oftarget droplets.

In some embodiments, detecting the first signal may comprise detectingthe first signal with a fast-response optical detector. Thefast-response optical detector may comprise a photo multiplier tube(PMT), a photodiode, or an avalanche photodiode detector (APD).

In some embodiments, the method may further comprise generating the dualfocusing, such as with an optical splitter or a birefringent polarizer.

In some embodiments, detecting the second signal may comprise detectingthe second signal with a camera.

In some embodiments, the method may further comprise illuminating thesecond point of detection with a laser or laser-like source. Thelaser-like source may be an LED.

In some embodiments, the second signal may be generated by stroboscopicillumination. The method may further comprise synchronizing dispensingand detecting the first signal or dispensing and detecting the secondsignal based on the first signal or the second signal.

In another aspect, a method for detecting droplets in bioassays isprovided. The method comprises providing a plurality of water-in-oildroplets to a first channel of a microfluidic device, wherein at leasttwo of the plurality of droplets each comprise at least one cell, atleast one particle, or at least one cell plus at least one particle, andwherein at least a portion of the first channel has a size of at least35 μm, for example at least about 60 μm, in each cross-sectional innerdimension; flowing the plurality of droplets past a first point ofdetection disposed along the first channel; directing laser energy tothe first point of detection, wherein directing comprises (1) passingthe laser energy through a laser modulator, the laser modulatorcomprising a remote focusing unit, an optical element generating anon-diffracting beam, or both, (2) directing the modulated laser energythrough a first objective or a second objective, wherein the firstobjective and second objective are disposed at an angle relative to oneanother, and (3) directing the modulated laser energy from the firstobjective or second objected through a prism adjacent to the firstchannel and onto the first point of detection, wherein the firstobjective and the second objective are disposed at a first corner and asecond corner, respectively, of the prism; detecting a first signal fromeach of the plurality of droplets as they flow past the first point ofdetection; and identifying target droplets based on the first signal.

In some embodiments, the method may further comprise sorting the targetdroplets from the rest of the plurality of droplets.

In some embodiments, the method may further comprise dispensing thetarget droplets.

In some embodiments, the angle may be about 60 degrees to about 120degrees. For example, the angle may be about 80 degrees to about 100degrees. Preferably, the angle may be about 90 degrees.

In some embodiments, directing modulated laser energy may comprisedirecting the modulated laser energy at an angle of about 30 degrees toabout 60 degrees relative to the first channel. For example, directingmodulated laser energy may comprise directing the modulated laser energyat an angle of about 40 degrees to about 50 degrees. Preferably,directing modulated laser energy may comprise directing the modulatedlaser energy at an angle of about 45 degrees.

In some embodiments, the remote focusing unit may comprise an electricallens or a tunable acoustic gradient (TAG) index lens.

In some embodiments, the optical element generating a non-diffractingbeam may comprise an axicon, an annular aperture, or a spatial lightmodulator.

In some embodiments, the prism may comprise a material having arefractive index of about 1.28 to about 1.6. For example, the prism maycomprise a material having a refractive index of about 1.29 to about1.58.

In some embodiments, the entire first channel may have a size of atleast 35 μm, for example at least about 60 μm, in each cross-sectionalinner dimension.

In another aspect, a method for detecting, sorting and dispensingdroplets is provided. The method comprises providing a plurality ofwater-in-oil droplets to a first channel of a microfluidic device,wherein at least two of the plurality of droplets each comprise at leastone cell, at least one particle, or at least one cell plus at least oneparticle; flowing the plurality of droplets past a first point ofoptical detection disposed along the first channel; detecting a firstsignal from each of the plurality of droplets at the first point ofdetection; identifying a first batch of target droplets based on thefirst signal; sorting the first batch of target droplets into a secondchannel of the microfluidic device; flowing the first batch of targetdroplets past a second point of detection disposed along the secondchannel; detecting a second signal from each of the first batch oftarget droplets at the second point of detection, wherein the secondsignal is generated by stroboscopic illumination; identifying a secondbatch of target droplets, the second point of detection being based onstroboscopic illumination; dispensing the second batch of targetdroplets; and indexing the second batch of target droplets such thateach dispensed droplet matches precisely the second signal detected fromeach of the second batch of target droplets.

In some embodiments, the stroboscopic illumination may be generated by aconstant or pulsed light source. For example, the stroboscopicillumination may be generated by modulating a continuous-wave (CW) lasereither directly or with an acousto optic modulator or by using a pulsedlaser source such as a q-switched laser.

In some embodiments, detecting the second signal may comprise detectingthe second signal with a camera.

In some embodiments, detecting the first signal may comprise detectingthe first signal with a fast-response optical detector. Thefast-response optical detector may comprise a photo multiplier tube(PMT), a photodiode, or an avalanche photodiode detector (APD).

In some embodiments, the first signal or second signal may comprise anoptical signal, an electrical signal, or an optical signal plus anelectrical signal. The method may further comprise comprisingsynchronizing dispensing and detecting the first signal or dispensingand detecting the second signal based on the first signal or the secondsignal.

In another aspect, a method for detecting, sorting and dispensingdroplets is provided. The method comprises providing a plurality ofwater-in-oil droplets to a first channel of a microfluidic device,wherein at least two of the plurality of droplets each comprise at leastone cell, at least one particle, or at least one cell plus at least oneparticle; flowing the plurality of droplets past a first point ofoptical detection disposed along the first channel; detecting a firstsignal from each of the plurality of droplets at the first point ofdetection, wherein the first signal is detected by a multi-zonedetection module comprising one or more detectors; identifying a firstbatch of target droplets based on the first signal; sorting the firstbatch of target droplets into a second channel of the microfluidicdevice; flowing the first batch of target droplets past a second pointof optical detection disposed along the second channel; detecting asecond signal from each of the first batch of target droplets at thesecond point of detection, wherein the second signal is detected by themulti-zone detection module; identifying a second batch of targetdroplets, the second point of detection being based on imaging; anddispensing the second batch of target droplets.

In some embodiments, the method may further comprise indexing eachtarget droplet of the dispensed second batch of target droplets with thefirst signal of the same target droplet.

In some embodiments, the one or more detectors may comprise a multi-zoneoptical detector with a single detection area in the microfluidic devicecomprising the first point of optical detection and the second point ofoptical detection. In some embodiments, at least a portion of the firstchannel or second channel disposed between the first point of opticaldetection and the second point of optical detection may comprise alooping channel that loops from the first point of optical detection tothe second point of optical detection. In some embodiments, themulti-zone optical detector may comprise a multi-channel photomultiplier or a camera.

These and other embodiments are described in further detail in thefollowing description related to the appended drawing figures.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates a schematic of a system for droplet generation (alsoreferred to herein as “encapsulation”), incubation, sorting, anddispensing in a microfluidic device, in accordance with embodiments. Thedispensing unit is optional and can be eliminated for applications thatonly require bulk sorting and collection of intra-droplet cells and/orentities.

FIG. 2 shows a schematic of a similar system to that shown in FIG. 1,except that the encapsulation unit is removed, in accordance withembodiments. A separate microfluidic chip or capillary-based platformmay be used for encapsulation.

FIG. 3 shows a schematic of a similar system to those shown in FIGS. 1and 2, except that the encapsulation and incubation units are removed,which can be done in combination or alone on separate microfluidic chipsor with microfluidic tubing (e.g., capillary) -based platform(s), inaccordance with embodiments. As an alternative configuration, amicrofluidic chip similar to FIG. 1 in which only the incubation unit isremoved can be used, for example for assay chemistries that areinstantaneous so the droplets can be sorted directly downstream withoutthe need of a separate incubation unit.

FIGS. 4A-4B shows schematics demonstrating the concept and advantages ofincluding laser modulation in a droplet detection unit, in part by usingremote focusing (RF), in accordance with embodiments. For example, atunable acoustic gradient (TAG) index lens (FIG. 4A), an electricallens, or other non-diffracting illumination schemes (FIG. 4B) such asBessel beams or Airy beams, can be used in conjunction with theobjective unit as a point(s) of detection of any of the systemsdescribed herein, in accordance with embodiments.

FIGS. 5A-5C shows an advanced optical configuration as part of a pointof detection, using two objectives positioned at an about 60 to about120-degree angle to each other at two corners of a prism, in accordancewith embodiments.

FIGS. 6A-6C illustrates an advanced optical configuration as part of apoint of detection, using two objectives positioned at an angel of about60 to about 120-degree to each other at two corners of a prism, in a waysimilar to that in FIGS. 5A-5C but further integrated with a remotefocusing device as illustrated in FIG. 4, in accordance withembodiments.

FIGS. 7A-7B illustrates an optical configuration for imaging fast movingtargets in a microfluidic system without motion blur, in accordance withembodiments.

FIGS. 8A-8F illustrate a channel comprising a single, pair(s), orarray(s) of permanent and/or tunable magnets with different shape, size,geometry, power, or arrangement at or before a point of detection forapplications where magnetic particles are encapsulated with one or morecells in droplets for assays, in accordance with embodiments. Differentdesigns can be implemented as illustrated in FIGS. 8A-8F.

FIG. 9 shows a schematic of a system for performing a two-step serialsorting on a microfluidic device, in accordance with embodiments.

FIG. 10 shows a schematic of a system for serial sorting as shown inFIG. 9 while a parallel detection design is implemented using adetection module with two or more zones, in accordance with embodiments.A dispensing unit can be added as an optional downstream unit, inaccordance with embodiments.

FIG. 11 illustrates an optical configuration with multi-channeldetectors such as multi-anode photomultiplier tubes (PMTs) or a camerafor simultaneous detection of droplets in two or more parallelmicrofluidic channels, like the one shown in FIG. 10, in accordance withembodiments.

FIG. 12 shows a schematic of a system having multiple points ofdetection (e.g., two, three, or more) such as PMTs or cameras fordroplet migration time determination and subsequent synchronization fromdroplet detection to dispensing, in accordance with embodiments. Eachdispensed droplet may be tracked and indexed to match the signal datathat are collected at one or more optical detection points, inaccordance with embodiments.

FIGS. 13A-13B shown exemplary design schematics of systems having one ormore non-optical (FIG. 13A) and/or optical (FIG. 13B) sensors for moreprecise tracking of droplets during sorting and dispensing, inaccordance with embodiments.

FIG. 14 shows a flow chart depicting a general exemplary workflow withprocesses to detect, sort, and dispense droplets by following one ormore modules and concepts described herein, in accordance withembodiments.

FIG. 15 shows various exemplary flow charts to methods for processingdroplet sorting, dispensing, and indexing, in accordance withembodiments.

FIG. 16 shows a schematic of an optical configuration for multi-pointdetection where the first and second points of detection are integratedinto a single field of view and the emission light is split and sent totwo downstream detectors, respectively, in accordance with embodiments.

FIGS. 17A-17B show schematics of systems comprising a section with oneor more bypass channels (i.e., “buffer zone”) to reduce the speed ofmobile droplets for imaging by using a camera, in accordance withembodiments.

FIG. 18 shows a schematic of an exemplary optical detector with a dualfocusing feature, as part of a point of detection, in accordance withembodiments.

FIGS. 19A-19D shows an exemplary implementation of an advanced opticalconfiguration as part of a point of detection, using two objectivespositioned at a 45-degree angle to each other at two corners of a prism(FIGS. 19A and 19C) or at a 45-degree angle without a prism (FIGS. 19Band 19D), in accordance with embodiments.

FIGS. 20A-20B show an exemplary implementation of a dual focusingfeature at a point of detection, in accordance with embodiments.

FIG. 21A shows an exemplary implementation of the design in FIG. 16 fora multi-point detection wherein the first and second points of detectionare integrated into a single field of view, in accordance withembodiments.

FIG. 21B shows exemplary signals detected using the system of FIG. 21A,in accordance with embodiments.

FIG. 22 shows an example of droplet imaging as part of a point ofdetection utilizing a buffer zone design as shown in FIG. 17B, inaccordance with embodiments.

FIG. 23 shows an exemplary implementation of droplet detection andindexing, in accordance with embodiments.

FIG. 24A shows an exemplary assembly of an exemplary optical sensor andits implementation to detect individual droplets, droplet size, dropletspeed, and droplet position along a flow channel, in accordance withembodiments.

FIGS. 24B-24C show exemplary signals detected using the system of FIG.24A, in accordance with embodiments.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying figures, which form a part hereof. The drawings illustrateembodiments of the present disclosure and, together with the detaileddescription, serve to explain the principles of the present disclosure.The drawings may not necessarily be in scale so as to better presentcertain features of the illustrated subject matter. In the figures,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, figures, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the scope of the subject matter presented herein.It will be readily understood that the aspects of the presentdisclosure, as generally described herein, and illustrated in thefigures, can be arranged, substituted, combined, separated, and designedin a wide variety of different configurations, all of which areexplicitly contemplated herein.

Although certain embodiments and examples are disclosed below, inventivesubject matter extends beyond the specifically disclosed embodiments toother alternative embodiments and/or uses, and to modifications andequivalents thereof. Thus, the scope of the claims appended hereto isnot limited by any of the particular embodiments described below. Forexample, in any method or process disclosed herein, the acts oroperations of the method or process may be performed in any suitablesequence and are not necessarily limited to any particular disclosedsequence. Various operations may be described as multiple discreteoperations in turn, in a manner that may be helpful in understandingcertain embodiments, however, the order of description should not beconstrued to imply that these operations are order dependent.Additionally, the structures, systems, and/or devices described hereinmay be embodied as integrated components or as separate components.

For purposes of comparing various embodiments, certain aspects andadvantages of these embodiments are described. Not necessarily all suchaspects or advantages are achieved by any particular embodiment. Thus,for example, various embodiments may be carried out in a manner thatachieves or optimizes one advantage or group of advantages as taughtherein without necessarily achieving other aspects or advantages as mayalso be taught or suggested herein.

Overview

Provided herein are systems, modules, units, and methods to detect,sort, and dispense a plurality of water-in-oil droplets in amicrofluidic device for various chemical and biological assays includingimmunotherapeutic screenings. In some embodiments, the droplets maycomprise at least a cell, at least a particle, or at least a cell plusat least a particle. The at least one cell may be provided from abiological sample such as tissue cells, immune cells, and/or engineeredcell libraries. Those cells may comprise low-abundance target cells,generally of ≤1%, <0.1%, or even <0.01% of a provided total cellpopulation.

The systems and methods provided herein may provide rapid,high-throughput, multiplexable genetic, protein, and other cellularanalyses down to single-molecule or single-cell level and can be usedfor several applications including but not limited to the isolation anddetection of immune cells, circulating tumor cells (CTCs), cell-freenucleic acids and exosomes, cancer initiating cells, cell druginteraction and resistance, cell-cell communication in tumormicroenvironments, and the analysis of genomes and epigenomes usingsingle-molecule next-generation sequencing technologies.

In one aspect, systems, module, units, and methods provided herein canbe used for the discovery of immunotherapeutics, specifically,bispecific antibodies (BsABs). The BsABs are unnatural biologics thatare engineered to recognize two different epitopes either on the same ordifferent target antigens. One of the exemplary applications can befocusing on T cell activating BsABs (TABs), as they are currently themost represented sub-class of BsABs, though the provided system andmethod can be applied to almost any BsAB formats. The method may employa droplet microfluidic-based system to compartmentalize and interrogateindividual BsAB-producing cells (optionally also expressing targetantigens) with co-encapsulated T cell reporters. Functional BsAB clonesmay be able to crosslink the T cell with antigen-expressing cell in thedroplet and activate T cell reporter to produce fluorescence, which inturn may allow “positive” droplets to be detected and sorted from aheterogeneous population.

In one aspect, one, two, or more points of detection may be used, thepoints of detection comprising at least one point of optical detectionthat is based on at least one laser or at least one laser-like source.In some embodiments, the laser may be provided through a unique opticalconfiguration that comprises a remote focusing module (e.g., a tunableacoustic gradient (TAG) index lens), and two objectives provided at anangle of about 60 to about 120 degree, or of about 90 degree, at twocorners of a prism, to provide an optical focal plane that crossespassing droplets in a channel of a microfluidic device at a point ofdetection. In some embodiments, a channel of at least 35 μm, for exampleat least about 60 μm, in any cross-sectional inner dimension may beprovided in a microfluidic device, to enable optical detection ofpassing droplets in the channel without constricting the droplets thatmay otherwise conventionally be constricted when using a channelgenerally narrower than about 35 μm or about 40 μm or about 50 μm. Forexample, the channel may be within a range of about 35 μm to about 200μm in any cross-sectional inner dimension, preferentially from about 40μm to about 120 μm. In some embodiments, the channel may be within arange of about 60 μm to about 200 μm in any cross-sectional innerdimension, preferentially from about 60 μm to about 150 μm.Alternatively, or in combination, the provided laser may be modulated tobe a non-diffracting beam. A non-diffracting beam may be achieved byusing an optical device such as an axicon lens, an annular aperture, aspatial light modulator, or the like, or any combination thereof In someembodiments, the prism may be made of a material with a refractive indexof about 1.28 to about 1.6, or of about 1.29 to about 1.57.

In some embodiments, a detector may comprise a sweeping mirror and/orrepetitive short illumination such as stroboscopic illumination in orderto effectively remove image blurring due to fast-moving objects such asa fast-flowing droplet and its entities contained therein. In someembodiments, a detector may comprise an optical detector that is coupledwith a magnet or a source of magnetic force to improve the opticalfocusing of magnetic or paramagnetic particles comprised by a passingdroplet at or prior to a point of detection in a channel of a microchip.

In another aspect, the provided system can deliver parallel detection ofpassing droplets, with a single multi-zone detection module comprisingtwo or more parallel channels in a microfluidic device.

In yet another aspect, droplet tracking and/or indexing may be providedby at least one detector or by at least one sensor. Tracking and/orindexing may in part be based on optical signals and/or non-opticalsignals such as contact or contactless conductivity, impedance, and/ormagnetic force. In some embodiments, at least one detector plus at leastone sensor may be used to provide data to track or index a passingdroplet, from an upstream point of detection to a downstream point ofdispensing along the flow direction in a channel of a microfluidicdevice. In some embodiments, entities in the droplet, such as cells andparticles, may be provided with a fluorescent tag to enable opticaldetection. In some embodiments, a point of droplet sorting may beimplemented immediately following a point of detection. In someembodiments, at least one detector and at least one sensor mayoptionally be implemented in a tandem manner, immediately following apoint of sorting but preceding a point of dispensing. In someembodiments, the at least one sensor may be an optical sensor, anon-optical sensor, or the combination or implementation of both.

In yet another aspect, a final point of detection may be implementedalong a flow channel of a microfluidic device at about 1 to about 60 cmupstream of a dispensing nozzle of a dispensing module. In someembodiments, the sorted droplets may be dispensed by a dispensing moduleinto collection tubes or plates, such as a 96-well plate, a 384-wellplate, or a multi-well plate or platform, in a controlled manner. Insome embodiments, a dispensed droplet may be provided with an index tomatch the dispensed droplet precisely with the collected data thatreflects an optical signal of a droplet that is detected at an upstreampoint of optical detection.

Provided are methods and processes for detecting, sorting, anddispensing droplets, in part using systems, modules, and units describedherein. In some embodiments, the process may comprise: providing in amicrofluidic device a plurality of water-in-oil droplets, at least somedroplets each comprising at least one cell, at least one particle, or atleast one cell plus at least one particle; passing and detecting thedroplets through a first point of laser-based optical detection along achannel of the microfluidic device to identify a first batch of targetdroplets; sorting a first batch of target droplets through a sortingactuator to obtain sorted droplets; detecting sorted droplets through asecond point of detection along a channel of the microfluidic device toidentify a second batch of target droplets; and dispensing a secondbatch of target droplets individually. Each dispensed droplet may beindexed in a manner such that each indexed dispensed droplet matchesprecisely with the collected data that reflects the optical signal of adroplet that is detected at an upstream point of detection.

In some embodiments, detecting droplets through a first point oflaser-based optical detection may comprise: (1) either one or morelasers modulated by a remote focusing unit, an optical elementgenerating a non-diffracting beam, or both; (2) a prism that ispositioned either above, below, or next to a channel of a microfluidicdevice, the channel having a size of at least 35 μm, for example atleast about 60 μm, in each cross-sectional inner dimension; (3) twoobjectives that are placed at an angle of about 60 to about 120 degree,or of about 90 degree to each other at two corners of the prism, whereinat least one of the objectives is used to provide a modulated laser toilluminate droplets that pass through the channel in the microfluidicdevice.

In some embodiments, particles provided in a droplet may bemicroparticle or nanoparticles of various shape or geometry, with a sizeranging from about 30 nanometers (nm) to about 30 micrometers (μm), orfrom about 100 nm to about 15 μm. The particles can be made of differentmaterials and can be labelled with molecules including proteins,antibodies, and functional chemical groups.

In some embodiments, droplet movement (also referred to herein as flow)in a microfluidic device may be driven by pressure generated by pumps orother pressure controllers. In some embodiment, droplet velocity mayrange from about 1 to about 900 mm/s, corresponding to a sortingfrequency of about 30 Hz to about 2000 Hz or higher.

In some embodiments, a provided system may comprise optional units fordroplet generation and/or incubation, which can be performed on a sameor separate microfluidic device prior to a point of detection. In someembodiments, the droplets can be incubated off-chip in a separatecontainer. In some embodiments, the system may provide dispenseddroplets that are indexed so that the identity of the dispensed dropletcan be correlated precisely with the corresponding data for thatindividual droplet. Those data may be collected at one or more points ofdetection prior to the dispensing step, in order to enable a morecomprehensive downstream post-processing analysis.

In another aspect, a sorted batch of droplets from a sorting point canbe provided to go through another step of sorting (i.e., serial sorting)to increase the purity of the final target droplets, which may beimportant for applications with a very complex biological startingsample comprising low abundance target events. In some embodiments, suchserial sorting may be used for sorting droplets containing entities orassays with different properties in multiplex assays. In someembodiments, a second sorting unit can be configured in a manner thatthe first and second points of detection will be in a same field of view(about 0.5 mm-about 5 mm) of a detector that is based on a multi-channeldetection module such as a multi-channel photo multiplier tubes (PMTs)or camera (or a camera-like device).

In some embodiments, in order to overcome motion blur of a fast movingtarget, the motion of a target (e.g., particle or cell) in a travelingdroplet can be compensated for by moving the image of the target at thesame speed during a longer camera exposure cycle, for example, byaddition of a movable (sweeping) deflector into the detection pathconsisting of an objective lens and a tube lens. To trigger imagedeflection at the appropriate time, a particle detector/sensor may bealso added upstream of the imaging device. Upstream particle detectioncan be achieved in several ways including but not limited to optical,electrical, and magnetic detection.

In some embodiments, a short (i.e., brief) illumination can be used toimage a moving target to overcome motion blur. The illumination must beshort enough in a way to ensure that the target moves less than thedesired spatial resolution (e.g., less than 10 μs for 1 μm spatialresolution at 100 mm/second (mm/s) flow speed). In some embodiments,each duration of short illumination (e.g., stroboscopic illumination)for one imaging frame can last about 0.5 milliseconds (ms) to about 50ms, of about 0.5 ms to about 10 ms, of about 5 ms to about 20 ms, about10 ms to about 30 ms, or about 20 to about 50 ms. In some embodiments,the short illumination is provided as a modulated or pulsed lightsource. In some embodiments, the modulation can be a short single pulseor a burst of pulses (e.g., 1-1,000 pulses per camera exposure cycle),where each repetitive pulse has a duration lasting about 1 nanosecond(ns) to 1 ms, about 1 ns to 99 ns, about 50 ns to 500 ns, about 200 nsto about 999 ns, or about 400 ns to 1 ms. For example, one kind ofrepetitive short illumination, namely stroboscopic illumination may begenerated by modulating a continuous-wave (CW) laser, either directly orwith an acousto-optic or electro-optic modulator, or by using the pulsesof a q-switched or mode-locked laser. The short illumination may besynchronized with a camera detector that detects moving droplets.

In some embodiments, motion blur of droplet image can be minimized orreduced by slowing down droplet moving in an image detection zone (i.e.,a “buffer zone”) in a device. The buffer zone may comprise of one ormore bypass channels that are connected to a main microfluidic channel,such that the fluid in the main fluidic channel will partially enter thebypass channel(s) to effectively reduce the droplet moving speed,thereby reducing the motion blur of droplet imaging. In someembodiments, two or more pillars can be provided at the interfacebetween the main fluidic channel and the bypass channels to constrainthe droplets moving along the main channel. In some embodiments, thebuffer zone may comprise a widened segment of a main fluidic channel. Insome embodiments, the buffer zone may comprise one or more compartmentsor chambers that are connected to a main fluidic channel through abypass channel. Creating a buffer zone may be combined with repetitiveshort illumination for enhanced suppression of motion blur.

In some embodiments, to capture multiple focal planes corresponding todifferent axial positions of a droplet at a point of detection with acamera detector, an optical device for remote focusing can be used andsynchronized with the camera exposure cycle and optionally with anillumination source modulation. Examples of remote focusing devices areTAG (tunable acoustic gradient) lenses and electrically tunable lenses(ETLs; e.g., Optotune Switzerland AG). The remote focusing device may besynchronized to take multiple images of a same droplet at differentfocal depths within different images or at multiple focal depthsoverlaid within the same image. Based on these images, beads and cellsoccurring at different axial positions within the same droplet can becaptured with a better focus.

Illustrative Embodiments

In some embodiments, a system 100 as shown in FIG. 1 may comprise amicrochip (i.e., microfluidic device) 105 with encapsulation unit 101,incubation unit 102, sorting unit 103, and a downstream microfluidictubing (capillary) -based dispensing unit 104. In the encapsulation unit101, one or more analytes 106 may be injected into a first inlet, andthe carrier oil, optionally mixed with surfactants, 107 may be injectedinto a second inlet with any types of pumps known to one of ordinaryskill in the art based on the teachings herein, such as syringe pumpsand pressure pumps, at a flow rate of about 1 to about 100 μL/min orhigher. Cells and/or particles may co-encapsulated into droplets 108.

As used herein, the terms “microfluidic device”, “microfluidic chip”,and “microchip” are often used interchangeably, which in general refersto a set of micro-channels etched or molded into a material (e.g.,glass, silicon, plastic, polymer, or polydimethylsiloxane), wherein themicro-channels forming the microfluidic chip are connected together inorder to achieve the desired features (e.g., mix, pump, sort, control ofbiochemical environment, etc.). It is understood that a person skilledin the art may readily fabricate such microfluidic devices in a properlyequipped mechanical or biomedical engineering lab or amicro-electro-mechanical systems (MEMS)/microfabrication core facility.

As used herein, the term “droplets” generally refers to a small amountof liquid surrounded by one or more immiscible or partially immiscibleliquid(s), also known as “emulsion.” Droplet volumes may range fromabout 0.01 nL to about 10 nL, and preferentially from about 0.02 toabout 2 nL for biological and chemical assays such as single cellanalysis. It is expected that a person skilled in the arts can readilyproduce the droplets with a syringe- or pressure-pump, a microfluidicchip with a flow-focus or T-junction feature, and/or a biocompatible oilsuch as 3M™ Novec-7500™ oil and fluorinert oil (FC-40), a stabilizingsurfactant such as PEG-PFPE tri-block or di-block co-polymers withconcentration ranging from about 0.5% w/w to about 3% w/w or higher, allof which are widely accessible in a properly equipped mechanical orbiomedical engineering lab or a MEMS/microfabrication core facility.

As used herein, the term “cells” generally refers to mammalian cellssuch as human and mouse cells, cancer cells, primary cells derived fromfresh tissues, immune cells such as B and T cells, non-mammalianvertebrate cells such as insect cells, yeast or fungal cells, bacterialcells, bacterial phages, hybrid cells, and any derivative or engineeredform of the cells thereof. It is understood that the cells can belabelled with a fluorescent dye such as FAM (carboxyfluorescein),Calcein AM, Green CMFDA, DRAQ7, Alexa Fluor series of dyes, and DyLightseries, and a fluorescent protein such as GFP (Green FluorescentProtein), YFP (Yellow Fluorescent Protein), EGFP, ZsGreen, mRFP (RedFluorescent Protein), and mCherry, and a fluorogenic enzyme substrate.

As used herein, the term “particles” is often used interchangeably with“bead” or “particulate objects” or “particulate entities”, which ingeneral refer to solid or soft-solid objects with a dimension scaleranging from nanometer (“nanoparticle”) to micrometer (“microparticle”),which may exhibit a shape or geometry reflecting a sphere, a cylinder, atube, a rod, an ellipsoid, and/or a branched configuration. Theparticles can be selected from a group consisting of organic andinorganic microbeads, polystyrene or plastic or glass beads,microspheres, silicon beads, nanoparticles, quantum dots, magnetic orparamagnetic beads, agarose gel, alginate microgel, and hydrogel, whichhave an equivalent diameter ranging from 10 nm to 50 μm, preferentiallyfrom 20 nm to 20 μm, and more preferentially from about 50 nm to about15 μm.

It is understood that particles are commonly available from a pluralityof commercial vendors such as Thermo Fisher, BD Biosciences, Bio-Rad,R&D Systems, BioLegend, Spherotech, and Abcam. Alternatively, theseparticulate objects can be made in a chemistry or material-science labby a person skilled in the art. It is further understood that particlesmay come as is, or pre-labelled with or functionalized for labelingwith: (1) fluorophores such as Alexa Fluor 405, FITC, GFP, and AlexaFluor 647; (2) affinity reagents such as a secondary antibody andProtein A; (3) an assay enzyme that may produce fluorescence orluminescence; (4) a chemical group; and/or (5) adaptor molecules such asBiotin and Streptavidin.

In some embodiments, as shown in FIG. 1, collected droplets may beincubated on-chip at Module 111 or off-chip for a predetermined durationof time depending on a specific assay. In some embodiments, theincubation Module 111 may comprise one or more environmental controlunits selected from a temperature control unit (with a preferredtemperature range of about 4° C.-98° C.), an oxygen control unit (with apreferred O2 level of about 0.01%-30%), a carbon dioxide control unit(with a preferred CO₂ level of about 0.1%-20%), and/or a humiditycontrol unit (with a preferred humidity level of about 50% to 99%).After incubation, the droplets may be reinjected into a microchip fordetection and sorting. Optionally, particles within the droplets may befocused for detection using a permanent or tunable electric magnet(single, pair, and/or array), or a combination of both (e.g., withModule 112), for enhanced focusing before a first point of detection113. In some embodiments, a first point of detection 113 can be based onoptical detection with an optical detector and a single-color laser beamor multi-color laser beams 114 in a case where a laser inducedfluorescence (LIF) method of detection is used.

As used herein, a point of detection generally refers to a locationwithin a channel of the microfluidic device corresponding to the focusor within the range of a detection module comprising a detector andoptional accessory components. The detection module may be capable ofdetecting a cell, a particle, and/or an assay readout signal within adroplet confined in a certain space (e.g., a fragment of a channel orreservoir) of a microfluidic device. Detection may be quantitative orsemi-quantitative. In some embodiments, the detection module may be anoptical detector, and the accessary components can be selected from anobjective, a mirror, a reflector, a lens, and a light source such as alaser, a xenon light, and/or a light-emitting diode (LED). Exemplaryoptical detectors include photo multiplier tubes (PMTs), camera-likedevices, charge coupled device (CCD) cameras, photodiodes, complementarymetal-oxide semiconductor (CMOS) cameras, and/or avalanche photodiodedetectors (APDs). In some embodiments, the detection module may be basedon a non-optical detector.

In general, a first point of detection 113 located immediately upstreamof, (i.e., preceding) a sorting junction along the flow direction, maycomprise a fast-response detector. Example fast-response detectorsinclude optical detectors such as a PMT, a photodiode, and/or an APD.

In some embodiments, detection signals may be sent to a data acquisition& processing unit 126 for signal processing. Upon detecting a signalindicative of a positive droplet (i.e., “target droplet”), theacquisition and processing unit 126 may deliver a trigger signal to asorting controller 125. The sorting controller 125 may then activate thesorting actuator 115 to redirect a moving target droplet 117 to a targetcollection channel in the microfluidic device. When the sorting actuator115 is not triggered, the moving droplets in the channel may continuetheir motion and enter the waste channel 116. In the meantime, the datacollected at an acquisition and processing unit 126 may optionally besent to a computer 127 for storage and further analysis.

As used herein, the term “data acquisition and processing unit” is oftenused interchangeably with “processor”, “processing unit”, or “processingchip”, which generally refers to an electronic circuitry and/or devicethat may carry out the instructions of a computer program by performingthe basic arithmetic, logic, controlling, and input/output operationsspecified by the instructions. The basic operations of a processor mayinclude, but are not limited to, the processing of collected samplesignals and converting the resulting signals into digital numeric valuesthat can be manipulated by a computer. A processor may send instructionsto other system units and interfaces (e.g., a sorting controller unit)to initiate a process (e.g., to activate sorting actuator). Exemplaryprocessors are Central Processing Units (CPUs), Field Programmable GateArrays (FPGAs), microprocessor (a central processing units contained ona single integrated circuit (IC)), Application Specific Instruction SetProcessor (ASIP; a component used in system-on-a-chip design), anddigital signal processor (I)SP; a specialized microprocessor designedspecifically for digital signal processing).

In some embodiments, droplet sorting may be performed at a sortingjunction, or a point of sorting on a microfluidic device by a sortingmodule; such a sorting module can be based on a dielectrophoretic (DEP),an acoustic, a piezoelectric, a microvalve-based, a dynamic streamdeflection based, and/or an electrical capacitance-based mechanism, in amanner that may be synchronized with an immediate upstream point ofdetection, which may be generally controlled by a data acquisition &processing unit.

In some embodiments, the sorted target droplets may be directed into amicrofluidic tubing (or channel or capillary tube) 119 through anadapter 118. The adapter can be plastic tubing or any other adaptiveconnectors known to one of ordinary skill in the art based on theteachings herein with an outer diameter (OD) ranging from about 0.1 toabout 5 mm. The microfluidic tubing (e.g., capillary), which is made ofglass, polymers, or any other materials with an inner diameter (ID) ofabout 0.02 to 1.5 mm, preferentially from 0.05 to 0.15 mm, can bepristine or coated.

In some embodiments, a second point of detection 121 may be used toverify that the target droplet has been sorted. Alternatively, or incombination, in some applications the second point of detection 121 maybe used to extract and/or to provide additional information from sorteddroplets, such as spatial fluorescence distribution within a cell.Additionally, the second point of detection 121 may also work inconjunction with data acquisition and processing unit 126 to preciselytrigger the dispensing Module 122 to dispense the sorted droplets.Similar to the first point of detection 113, a laser or light source 120can be used for illuminating the second point of detection to generatethe signal detected by the detector associated with the second point ofdetection 121. The dispensing Module 122 with nozzle 123 can comprise anx-y-z moving stage or a rotating moving stage configured to move nozzle123 to dispensing collector 124 (e.g., to a specific well of amulti-well plate collector 124). The dispensing collector 124 can be a96-well plate, 384-well plate, 1536-well plate, or any other multi-wellplates, PCR tubes, PCR strips, or any array of interest.

FIG. 2 depicts a system 150 which has a similar setup as in system 100of FIG. 1, except that the droplets are generated off chip. The system150 may comprise a microchip 154 comprising an incubation unit 151, asorting unit 152 downstream of the incubation unit 151, and a dispensingunit 153 downstream of the sorting unit 152, which may be substantiallysimilar to those described herein, e.g., including first and secondpoints of detection 162 and 170, respectively, a sorting actuator 164,first and second channels connected to one another by a sortingjunction, etc. The droplets may be generated on a separate microfluidicencapsulation chip that, in principle, will function similar to theencapsulation unit 101 described in FIG. 1. The droplets may then beprovided into a channel of a microchip 154, e.g., through a pressurizedmechanism, for subsequent on-chip incubation, detection, sorting, and/ordispensing as described herein. In some instances, it may be beneficialto generate the droplets off-chip (e.g., on a separate microfluidicencapsulation chip). Depending on assay types and applications,different settings and operation conditions may be required for optimaldroplet generation to enable efficient downstream processes such asdroplet sorting, which may not be achievable or practical on a fullyintegrated multifunctional microfluidic chip.

FIG. 3 shows a system 190 which has a similar setup as in system 150,except that the droplets are also incubated (or not) off-chip. Thesystem 190 may comprise a microchip 193 comprising a sorting unit 191downstream of the channel inlet and a dispensing unit 192 downstream ofthe sorting unit 191, which may be substantially similar to thosedescribed herein, e.g., including first and second points of detection199 and 206, respectively, a sorting actuator 200, first and secondchannels connected to one another by a sorting junction, etc. Thedroplets may be incubated in a substantially similar manner to theon-chip incubation described herein. The droplets may then be provided(e.g., pipetted, injected, etc.) into a microchip 193, e.g., through apressurized mechanism, for subsequent on-chip detection, sorting, anddispensing as described herein.

In some embodiments, as illustrated in the systems 100, 150, and 190(FIGS. 1-3), particles encapsulated in droplets may optionally befocused by magnetic force exerted by a permanent or tunable electricmagnet (e.g., a single magnet, a pair(s) or magnet, and/or an array ofmagnets) for enhanced focusing before the first point of detection(e.g., first point of detection 113, 163, or 119, respectively), inwhich case a single-color laser beam or multiple-color laser beams maybe used as an excitation source for laser-induced fluorescencedetection.

Many targets of interest (e.g., cells) are low abundance or rare eventsin complex biological samples. For instance, it is generally understoodthat antigen-specific primary B cells often account for <0.1% or <0.01%of a B cell immune repertoire, and an antigen-specific primary T cellcan be present at lower than 0.01% or 0.001% of a T cell immunerepertoire. As used herein, the term “low abundance” or low-abundance”generally refers to any incidence that is lower than about 1%. As usedherein, the term “rare” generally refers to any incidence that is lowerthan about 0.1%.

Moreover, common biological samples such as those derived from blood orother tissues are often very complex, which may be associated with ahigh background signal, making any screening assay a daunting task. Toeffectively determine and isolate the low abundance or rare events byusing droplet-based assays, the assay system should perform with bothhigh sensitivity and specificity. An optical detection module, ifincluded in the system, may provide high signal-to-noise (SNR) andsignal-to-background (SBR) ratios. Therefore, a uniformly high spatialresolution may be required. At the same time, to be able to process alarge amount of sample such as millions of B cells, T cells, or othertypes of cells, the temporal resolution (i.e., detection speed) shouldbe high. However, to achieve both high temporal and high spatialresolution can be challenging.

A common approach to achieving high temporal resolution is the use ofsingle point photo multiplier tubes (PMTs). PMTs are highly sensitiveoptical detectors that can provide photon count rates in excess of 10million counts/second, thereby allowing for high sample rates in excessof 1 million samples/second. Meanwhile, a widely used method forillumination in a fluidic channel is to focus a laser beam with acylindrical optical element in order to illuminate the channel with athin sheet of light. Hence, the minimum object size that can be resolvedcorresponds to the thickness of the light sheet, which is in turndetermined by the numerical aperture (NA) of the focusing lens. Besidesthe extension of the focal spot, the lens' NA value also controls thedepth of focus/confocal parameter. The confocal parameter defines howrapidly defocusing occurs with increasing distance from the focal planeof the lens. Unfortunately, both parameters, the size of the focal spotand the depth of focus, are related to the NA such that with increasingNA value the focal spot gets smaller while defocusing occurs morerapidly. Particles passing the laser sheet at the focal plane may bedetected with high spatial resolution while those passing at theperiphery of the beam may have a broadened signal profile. Hence,conventional sheet illumination provides a compromise between themaximum achievable spatial resolution in the focal plane and the depthof field over which this resolution can be achieved.

To overcome the issue described in the foregoing paragraph, theinventors have devised a novel strategy using remote focusing (i.e.,“re-focusing” or “RF”) of the illumination beam to effectively create athin homogeneous illumination profile and provide a detection efficiencythat is independent of the axial position, as illustrated in FIG. 4A(Module 300). The use of RF may be advantageous as it can increase thedepth of field when compared to regular objectives and can provide auser-specified changeable focal length with sub-microsecond temporalresolution. Alternatively, or in combination, non-diffracting beams suchas Bessel or Airy beams can be used as illustrated in FIG. 4B (Module310).

Achieving a homogeneous high spatial resolution without any compromisesin temporal resolution is highly desired for applications that involvedetecting and sorting low-abundance events from a large amount ofheterogeneous or complex starting samples. In the following description,an example is provided as a droplet-based cell sorting application. Itshall be noted that the benefit of the methods, devices, and systemsdescribed herein are not meant to be limited to this particularapplication but instead may be universally applicable. This specificexample is to help illustrate the basic principle of the system proposedherein.

For antibody discovery, antibody producing cells, such as primary Bcells, can be prepared as single cell suspension from spleen or bonemarrows of an immunized animal by following well-established protocols.These antibody producing cells can be encapsulated in droplets asdescribed herein together with fluorescently-labelled antigens (i.e.,“dyed antigen”) that can bind to antigen-specific antibodies (i.e.,“antibody of interest”) that are secreted from an encapsulated B cell.However, in at least some instances the labeled antigen may behomogeneously distributed throughout the droplet such that thefluorescence signal will be the same independent of the presence orabsence of the antibody of interest. This issue can be overcome byco-encapsulating a microsphere with a functionalized surface that canspecifically anchor the antibodies released from a co-encapsulated cell,e.g., by an IgG affinity reagent such as Protein-A and anti-IgGantibodies. The microsphere may capture the antibodies of interest,which in turn may capture fluorescently-labeled antigens, therebyleading to fluorescent focus formation on the microsphere. Thefluorescent focus can be optically detected as an assay readout of apositive droplet (i.e., “target droplet”), which can be sorted anddispensed in a real-time or near real-time fashion. Two factors maydetermine the accuracy and efficiency of detecting optical signal ofsuch a fluorescent focus:

The spatial resolution should be sufficient to resolve a microspherewithin a droplet, for example a 5-μm microsphere within a 100-μmdroplet;

Out-of-focus signal should be sufficiently suppressed to separate thesignal localized to the microsphere from signal that can be attributedto unbound fluorescently-labelled antigens in the droplet.

Therefore, the illumination beam may be focused into a thin sheet togenerate sufficient spatial resolution to distinguish small featuressuch as a particle or a cell. Tight focusing, however, may lead togreater beam divergence, which in turn may cause a loss of spatialresolution above and below the focal plane. The detected signal may bethe sum of the unbound dyed antigen within the droplet and the dyedantigen bound to the microparticle via the anchored antibody ofinterest. If the droplet passes the point of detection with the particlenear the focal plane, the particle signal can be clearly resolved.However, if the particle within the droplet passes near the edge of thefocal point, the particle signal will be broad and of lower amplitude,which can be indistinguishable from the signal of the free dyed antibodywithin the droplet. Hence, a large number of false negative events maybe produced. To overcome this problem, extended depth of fieldillumination and detection can be used as described herein (e.g., inFIGS. 4A and 4B).

FIG. 4A illustrates the use of RF to modulate a beam of laser energy ata detection point of any of the systems described herein. FIG. 4Billustrates the use of NBD beams to modulate a beam of laser energy at adetection point of any of the systems described herein. In bothembodiments, a beam of light may be passed through a cylindrical opticalelement focusing the light into the back aperture of the objective lens(304/314) aimed at the sample, in this case delivered through a point ofdetection along a channel (302/312) of a microchip (301/311) comprisingsupporting substrate (303/313). In an exemplary embodiment, themicrochip 301/311 can be made of PDMS on a glass substrate 303/313.

Along the focusing axis of the cylinder lens, the initial beam may bede-magnified by the magnification factor of the objective lens given theappropriate focal length of the cylinder element. The initial beamdiameter, focal length of the cylinder lens, and/or magnification factorof the objective lens may be adjusted for the illumination to span thewidth of the fluidic channel at the sample plane. Along the non-focusingaxis of the cylinder lens, the parallel illumination beam may beunaltered until it passes the objective lens focusing the light into asheet at the sample.

The minimum sheet width and confocal parameter may be defined by the NAof the objective lens and the incident beam diameter, i.e., theeffective NA of the objective lens. Light from the focal plane of theobjective lens may be collected and separated from the illumination viaa beamsplitter and focused onto an aperture. The purpose of the apertureis to reject light from out-of-focus planes at the sample in order toreduce background noise. A rectangular aperture may be preferred over acircular one to better match the shape of the illumination profile whichis extended across the channel and focused along the channel. Withoutthe addition of further optical elements, several compromises must bemade in order to make this configuration work:

-   -   1. If a highly effective illumination NA is used, the light        sheet may be thin in the center of the fluidic channel, but        broad towards the edges of the channel;    -   2. By instead lowering the effective illumination NA, a more        homogeneous illumination profile may be created at the expense        of reduced spatial resolution;    -   3. A confocal aperture matching the light sheet's xy dimensions        after objective lens magnification may most effectively        suppresses background light from xy positions not directly        illuminated by the sheet. It may also suppress signal from axial        positions not near the focal plane of the objective lens        resulting in a loss of sensitivity in those regions; and    -   4. A larger confocal aperture may allow for the detection of        signals from the entire height of the channel but may result in        increased background.

The use of remote focusing (305 & 315), as shown in FIGS. 4A-4B, canovercome some or all of these compromises. By the introduction of a lenswith variable focal length before the objective lens interfacing withthe sample, the sample focal plane can be moved in axial direction, asshown in FIG. 4A. Hence, even if the illumination/detection is highlyconfined in axial direction, a large axial range such as the entirechannel height may be accessible via optical translation of the focus.Alternatively, non-diffracting beams can be used to generate asheet-like illumination with minimal divergence (315) as shown in FIG.4B. With either remote focusing setup, the previously contradictingparameter optimization scheme can work to maximize spatial resolution,depth of field, and high background noise suppression in the followingmanner:

-   -   1. A high effective illumination NA may be used to tightly focus        the illumination beam along the direction of the channel,        resulting in improved (e.g., maximum) spatial resolution at the        focal plane of the objective lens interfacing with the sample;    -   2. A confocal aperture matching the xy dimensions of the sheet        illumination may be used before the detector to suppress        background noise as well as low resolution signal from        out-of-focus planes where the sheet thickness is large;    -   3. With measures 1 and 2 above, we effectively confine our        detection to a small portion of the channel height. To detect        particles with the same spatial resolution and sensitivity, the        excitation/effective detection volume may be translated along        the optical axis via remote focusing; and    -   4. The translation may occur fast enough to not compromise        sampling speed. In a preferred embodiment, the channel should be        scanned at least once along its entire axial extension during        the time it takes one sample.

While many methods and optical modules are available for remote focusingin support of our proposed system and method, without limitation wefocus on the use of a TAG lens for its high speed to avoid compromisesin sampling rate, i.e., temporal resolution. At least two remotefocusing designs are described herein:

-   -   1. Module 300 shown in FIG. 4A: Remote focusing may be used to        extend the depth of the field of the optical detection for        homogeneous resolution and detection efficiency within the        entire channel height;    -   2. Module 310 shown in FIG. 4B: The use of non-diffracting beams        for a homogeneous illumination of the entire channel height for        homogeneous resolution and detection efficiency.

To screen for droplets (327, 347, 376, 399, 419, & 438) comprisingrelevant cells/molecules (328, 348, 349, 377, 378, 400, 420, 421, 439, &440) with high sensitivity and specificity, the optical detection set upshould provide high SNR and SBR ratios while maintaining a high temporalresolution for high throughput. Hence, uniformly high spatial resolutionin combination with high detection efficiency is desired. Besides thepresence of a particle or cell, the spatial position of a particle orcell within the screened volume may be of interest as well. In ahigh-throughput microfluidic assay system, this can be challenging.

As described herein, spatial modulation can be used to extractinformation about the size, shape, and/or position of a cell orparticle. The sample may be illuminated and/or light may be detectedwith a spatially modulated pattern. Depending on the particle or cell'ssize, shape, and/or position, an additional modulation is added to thesignal. By appropriate “de-modulation”, the size, shape, and/or positionof particle or cell, can be inferred. However, with this approachspatial information is encoded into the time domain, resulting in a lossof temporal resolution.

In an alternative embodiment to overcome this challenge, the modulesillustrated in FIGS. 5A-5C, with decoupled, perpendicular illuminationand detection to extract spatial information about the sample at highspeeds with the use of single-point detectors or cameras, may beutilized in any of the systems described herein. The illustrated schemesmay allow for detection of twice the signal intensity as the samecell/particle passing by two perpendicular optical planes as describedin other embodiments herein. By orienting the illumination/detectionplanes at about 45 degrees with respect to the channel direction, we mayovercome the limited focal depth which may be problematic in someinstances when illuminating/detecting perpendicular to the channel.Therefore, the modules may allow for the use of lenses of high NAs whilealso maintaining isotropic detection efficiency independent of thechannel height. The modules shown in FIGS. 5A-5C may also allow forcollection of more photons, and therefore detection of lower strengthsignals as well as identification of finer spatial features, when usedin combination with any of the systems described herein. The proposednew designs can be used for detecting one, two, or more cells and/orparticles inside a droplet (FIGS. 5A-5C). The new designs may furtherallow for detection of twice a given cell or particle inside a dropletthat crosses the two perpendicular optical planes (i.e., detection attwo different locations within the droplet and/or channel). Further, byorienting the illumination/detection planes at about 45 degrees withrespect to the channel direction, the limitation in the focal depth in aconventional optical setup, where the coupled illumination/detectionlight path is perpendicular to the channel direction, may be overcome.Therefore, the modules described in FIGS. 5A-5C may allow for use oflenses with a high NA while maintaining isotropic detection efficiencyindependent of the channel height, resulting in collecting more photonsand therefore detecting weaker signals as well as finer spatialfeatures.

Achieving a high and homogeneous spatial resolution without anycompromises in temporal resolution or detection efficiency is highlydemanded in applications that require detecting and sorting single cellsor particles. In addition, it is of utmost importance to collect as manysignal photons as possible to maximize signal strength and enhance theability to detect weak signals. For example, in antibody discovery, thepresence or absence of certain cellular or subcellular structures of anantibody producing cell can be a deciding factor on whether a cell ispositive or not. To get significant resolution on these structures, ahigh and isotropic spatial resolution is required.

The problem with the conventional, single objective lens-basedapproaches are: To maximize spatial resolution as well as detectionefficiency, a high NA lens is demanded;

-   -   1. High NA lenses have a limited depth of focus. While objects        near the focal plane will be clearly resolved, spatial features        of objects distant from the focal plane will appear blurred;    -   2. Cameras are of limited usefulness as detectors if the imaging        plane is parallel to the channel direction. To image the entire        channel's cross-section, the imaging plane needs to be oriented        at a certain angle.        Signals from out-of-focus planes are blurred, hence, spatial        features of objects at those out-of-focus locations cannot be        resolved.

In some embodiments as exemplified in FIGS. 5A-6C, a system is providedto overcome this problem and improve the sensitivity and accuracy ofdetecting intra-droplet cells and/or particles, the system comprisingdecoupled yet perpendicular excitation and detection that is positionedat an angle to the channel direction can be used as described in thefollowing paragraphs.

In the system, two objective lenses (322/324, 342/344, 371/373, 393/395,413/415 & 433/435) may be positioned perpendicularly ornear-perpendicularly to each other, wherein the excitation laser beamspassing through the objectives interface with a microfluidic channel atan angle of about 30-60 degrees, of about 40-50 degrees, or of about 45degrees. In the excitation path, a cylinder optical element may be usedto generate a sheet of light (330/331, 351/352, 379, 402/403, 423/424 &442) illuminating a cross-section of a microfluidic channel (329, 350,380, 401, 422 & 441). This cross-section may be imaged via an objectivelens onto a detector which can be a single-point detector such as a PMTor a multi-channel detector such as a camera. Hence, in the plane ofobservation, the spatial resolution may be defined by the NA of thedetection lens. Perpendicular to the observation plane, the spatialresolution may be determined by the thickness of the light sheet usedfor illumination, which in turn may depend on the NA of the excitationlens.

To minimize optical aberrations introduced by illuminating/detectingthrough materials with different refractive indexes, a prism (323, 343,372, 394, 414 & 434) may be used to rotate the interface. As areplacement for or in addition to the prism, refractive index matchingfluids (326, 346, 375, 398, 418 & 437) may be used to improve signalquality. Since the arrangement of the first and second objectives can besymmetric, either objective lens can serve for illumination ordetection. Hence, by introducing a beam splitter, a detector and a lightsource can be fitted to each arm, respectively. Particles and/or cellsinside a droplet passing the channel can be detected twice (321/325,341/345, 391/397, 411/417 & 431/436) in two perpendicular planes,respectively. If a particle or cell passes in the center of the channel,it may be detected in both arms at the same time. If it passes below orabove the center, it may be detected in the two arms at different times.Hence, from the time delay between the two detection arms, the spatialposition of the particle within the channel can be inferred with twodistinct single-point detectors. If a camera is used, images can bedirectly taken from the cross-section of the channel. Therefore, notonly the spatial position, but also an image of the particle passing theobservation plane can be obtained.

FIG. 5A shows detection of the same particle or cell as it moves throughthe channel at two different locations and at two different times. Boththe excitation and detection paths may use the same excitationwavelength and emission filter. FIG. 5B shows detection of differentparticles or cells with matching fluorophores at two different locationswithin the droplet. The two excitation/detection paths may use differentexcitation wavelengths and emission filters. FIG. 5C shows detection ofdifferent particles or cell with only a single excitation/detection pathin order to increase light throughput.

The proposed scheme can be used for applications with a dropletcomprising a single cell or a single particle as shown in FIG. 6A, orfor a droplet comprising two and more cells or particles as shown inFIGS. 6B and 6C. The prism may be used to optically interface with amicrofluidic chip to minimize optical aberrations at this angle. Indexmatching fluid can be used to maintain a constant refractive index. Thespatial position of cells and/or particles within the microfluidicchannel can be determined by the timing of the signal detected in bothillumination/detection arms. Achieving a high and homogeneous spatialresolution with minimal compromise in temporal resolution or detectionefficiency is in high demand in any given particle-counting orsorting-application based on optical detections. In addition, it may beimportant to collect as many signal photons as possible to maximizesignal strength, enhance the ability to detect weak signals, andultimately improve sensitivity and also provide a quantitative system.

In summary, the exemplary modules shown in FIGS. 5A-6C may provide oneor more of the following benefits, which can be integrated in any of thesystems described herein:

-   -   1. High NA lenses can be used without compromising the depth of        field, resulting in higher spatial resolution and higher        detection efficiency;    -   2. The detection efficiency along the observation plane may be        isotropic;    -   3. Light throughput may be improved (e.g., maximized) because no        aperture is used to suppress out-of-focus signals;    -   4. By using both objective lenses for illumination as well as        detection, the sample may be observed at two different planes        and the spatial position of the sample within the channel can be        calculated from the time delay between the two detection events;    -   5. Cameras can be used as detectors capturing images of a        cross-section of the channel. Hence, fine details of the sample        can be spatially resolved.

While the spatial resolution may already relatively uniform with theabove-described approaches, it can be further improved by providingremote focusing (392/396, 412/416 & 432) of the excitation beam in thesetup illustrated in FIGS. 5A-5C. To summarize, in a preferredembodiment we propose the integration and use of one or more of thefollowing modules in our systems:

Module 320: Each objective may generate a light sheet in the detectionplane of the opposing lens. By the delay of the detected signal in bothdetection modules, the axial particle position may be obtained;

Module 370: Multiple color channels may be used with the same objectivelens;

Module 340: Optionally, multiple detection channels may be split betweenthe two lenses to avoid crosstalk between or among different colorchannels;

Module 390: Remote focusing or non-diffracting beams may be used asdescribed herein for a more efficient and more quantitative particledetection;

Module 410: Optionally, with remote focusing or non-diffracting beams,multiple detection channels may be split between the two lenses to avoidcrosstalk between or among different color channels;

Module 430: Alternatively, with remote focusing or non-diffractingbeams, multiple color channels can be used with the same objective lens.

For high-throughput droplet detection and sorting applications, thedroplets can comprise intra-droplet objects (e.g., cells and/or beads)that move within the droplets. The relative position of these objects tothe optic focal plane at a point of detection can be random and as aresult may lead to poor focusing (i.e., poor signal/noise ratios), suchthat the detection efficiency for these moving objects may besuboptimal, particularly if the droplet diameter or the fluidic channelwidth is significantly larger than the focal plane height. In someembodiments, two laser beams can be used to provide dual focusing aspart of a point of detection to improve the detection efficiency ofintra-droplet moving objects. In some embodiments, a single laser beamcan be split into two laser beams using a fiber optical splitter oroptical element to provide dual focusing at a point of detection. Forinstance, a laser modulator comprising an optical device with abirefringent element can be used to efficiently provide a line-shapedlaser illumination through splitting the laser light into two separatelaser beams, thereby leading to dual focusing (e.g., Module 730 shown inFIG. 18). Such a birefringent element (i.e. birefringent polarizer) cansplit unpolarized light or light polarized at a 45-degree angle, byrefraction into two beams of linearly polarized light with orthogonal ornear orthogonal polarization. Given dual focusing, each droplet cantravel through two foci that are closely positioned one after anotheralong the droplet flow direction, thereby improving the probability thatat least one focus can yield optical signals representing intra-dropletobjects with improved signal-versus-noise profile. The distance betweenthe two foci can be tuned by adjusting the distance between theobjective and the optical device. Suitable birefringent polarizers arerepresented by Nicol prisms, Glan-Thompson prisms, Glan-Foucault prisms,Glan-Taylor prisms. Rochon prims, Senarmont prisms, and Wollaston prismsare other examples of birefringent polarizers consisting of twotriangular calcite prisms that are cemented together. Exemplaryimplementations of dual focusing by using an optical device comprising aWollaston prism, as part of a point of detection, are shown in FIGS.20A-20B.

For high-throughput droplet sorting and dispensing applications, thetypical flow rate can be as high as 100 mm/s, or in some cases, up toabout 900 mm/s. While it may be desirable to take an image of afast-moving target, camera frame rates are often too slow to capture thetargets (472 & 490) flowing through a channel (452 & 483) of a microchip(451 & 482) on a substrate (453 & 484) without motion blur. For example,a target spatial resolution of 1 μm would require an exposure time ofless than 10 μs at a flow rate of 100 mm/s to avoid motion blur.Otherwise, a particle (472 & 490) passing a camera's field of view (456,471, 487 & 494) during a single exposure cycle would appear as a streak(456 & 487). However, the motion of a particle, if known, can becompensated by moving the image of the target at the same speed during alonger camera exposure cycle. This can be achieved, for example, byaddition of a movable (“sweeping”) deflector (459) into the detectionpath comprising an objective lens (454, 460, 485 & 492) and a tube lens(455, 470, 486 & 493), as shown in FIG. 7A (Module 450). Suitabledevices include sweeping mirrors, acousto-optic deflectors, and spatiallight modulators.

For droplet detection in a microfluidic system, the speed of a targetdroplet can be determined through measuring the flow rate. To triggerimage deflection at the appropriate time, a sensor that senses or countsdroplets (457 & 491) may be added upstream of the imaging device.Upstream droplet sensing/counting by the sensor can be achieved inseveral ways as disclosed herein, which include but are not limited toan optical, an electrical, and a magnetic detection method. Spatial andtemporal resolution of the sensor should be sufficient to sense thepresence of droplets flowing at a speed of up to 900 mm/s.

As used herein, the term sensor generally refers to a module or devicethat detects and converts the physical parameter of a passing dropletinto a signal which can be measured electrically. The sensor can benon-optical, optical, or a combination of both, which senses or counts adroplet, droplet size, droplet, and/or relative position of the dropletwhen passing through the sensor's sensing area, regardless of whether itis a target or non-target droplet, i.e., the sensor isnon-discriminative for a target droplet versus a non-target droplet. Incomparison, the signal detected by a detector at a point of detection isdiscriminative in terms of the detector's ability to quantitatively orsemi-quantitatively detect a cell and/or a particle within a droplet.Exemplary sensors are described herein, which includes those illustratedin FIG. 7 and other exemplary systems disclosed herein, which has acomponent labelled as “sensor”.

Alternatively, a short illumination pulse 489 can be used to image themoving target without motion blur as shown in FIG. 7B (Module 480). Thepulse may be short enough that the target has moved less than thedesired spatial resolution. For example, at a flow speed of 100 mm/s anda desired resolution of 1 μm, the illumination duration should be lessthan 10 μs. In some embodiments, the short illumination (e.g.,stroboscopic illumination) for each imaging exposure cycle has a totalduration of about 0.5 ms to about 50 ms, of about 0.5 ms to about 10 ms,of about 5 ms to about 20 ms, about 10 ms to about 30 ms, or about 20 msto 50 ms. This can be achieved, for example, by using q-switched ormode-locked lasers (e.g., active q-switched lasers based onacousto-optic modulator (AOM) and/or electro-optic modulator (EOM)).High resolution images of targets may comprise valuable information,which can be used for subsequent sorting and dispensing, as well astracking that may facilitate the sorting and dispensing. In summary, thefollowing modules are proposed, which may optionally be integrated intoany of the systems described herein:

Module 450: To obtain high resolution images of targets moving though amicrofluidic device at high speeds, a sweeping deflector may be used inthe detection path to compensate for the target movement and obtain amotion artifact free image;

Module 480: To obtain high resolution images of targets moving though afluidic device at high speeds, stroboscopic illumination may be used inthe excitation path to avoid motion artifacts.

In some embodiments, to obtain high resolution images of targets movingthrough a fluidic device at high speeds, the traveling targets (e.g.droplets) can be slowed by providing a “buffer zone” along a fluidicchannel (e.g. a sorting channel) in a microfluidic device. The bufferzone may be provided with one or more bypass channels (e.g., side poresor side channels) that are connected to a main fluidic channel withtravelling droplets, such that the fluid in the main fluidic channel canpartially enter the bypass channels to effectively reduce the movementspeed of droplets, thereby reducing the motion blur during dropletimaging as part of a point of detection. In some embodiments, one or twoarrays of pillars may be provided at the interface between the mainfluidic channel and the bypass channels to constrain the travelingdroplets moving along the main fluidic channel (e.g., Modules 710 and720 shown in FIGS. 17A-17B). In some embodiments, the buffer zone maycomprise a widened fluidic channel (e.g., Module 710 shown in FIG. 17A).In some embodiments, the buffer zone may comprise one or more sidechambers that are connected to a main fluidic channel through a bypasschannel. In some embodiments, the bypass channels may be positioneddownstream of a sorting junction. In some embodiments, the bypasschannels may be positioned downstream of a sorting junction and upstreamof a dispensing nozzle. In some embodiments, the buffer zone with bypasschannels can be implemented at a point of detection, which areexemplified in FIGS. 17A-17B and FIG. 22. Creation of a buffer zone maybe combined with repetitive short illumination for enhanced results.

In some instances, it may be of great interest to have fast movingparticles inside microfluidic channels in the same plane for precisedetection and increased sensitivity, which in turn may improve theoverall sorting and dispensing efficiency. The improved sorting anddispensing may be critical for isolating low abundance or rare events inmany real-world applications, which typically favor maximal recovery ofall positive events and maximal removal of false positive ones.

In general, finding non-destructive methods to bring fast-movingdroplets in the field of view of the objectives have significant valuesin droplet microfluidics, where limitations exist in selecting shape,material, and dimension of a microchannel, as well as the selecting thesize of droplets. Magnetic forces are one solution to address this need,which can be implemented in any of the systems described herein.

In some embodiments, one, a pair, or array(s) of magnet(s) can be usedbefore a point of detection in combination with magnetic particle(s)that may be provided together with a cell in a droplet, as shown inFIGS. 8A-8F. Magnets can be permanent, tunable electric, or acombination of both, which can be made of different material withvarious shapes, geometries, dimensions, and powers. Also,magnetic-microparticles or nanoparticles are widely available formolecular and cell biology applications. Magnetic particles may beavailable in a form that is coated with various functional groups suchas carboxyl groups, proteins, and oligonucleotides, which may enable thespecific capture of desired target molecules, cell types, or organellesavailable in a given droplet. Various combinations of magnets can beimplemented into our systems, as exemplified in FIGS. 8A-8F.

In one embodiment shown in FIG. 8A, a magnet 505 may be placed at adistance of about 0 mm to about 20 mm from a nearest downstreamdetection point. Magnetic forces generated by magnet 505 may graduallyalter the path of magnetic particle(s) 401 provided in a droplet 502 asthey move toward the magnetic field in the microfluidic channel 504. Byadjusting the flow rate and/or magnetic forces per defined application,one should be able to concentrate magnetic particles (e.g., formingclusters) next to each other and near the detection side of themicrofluidic channel 504 within the field of view of the objective 507.The concentrated magnetic particles in turn may generatenear-homogeneous and strong signals. It should be noted that adonut-shaped magnet 505 can also be integrated into the objective 507,making the distance from a nearest downstream point of detection 0 mm oralmost 0 mm.

In the embodiment shown in FIG. 8B, an array of magnets 515 may beplaced at a distance of about 0 mm to about 20 mm from a nearestdownstream detection point. Magnetic forces generated by this array ofmagnets 515 may alter the path of magnetic particle(s) 511 inside thedroplet 512 as they move toward magnet area in the microfluidic channel514. This array design may be particularly useful for applications wherethere are multiple, larger, and/or high-density magnetic particles 511inside droplets 512, which otherwise may lead to particles exiting thedroplets if only one magnet is used. By optimizing the flow rate,magnetic forces, and/or array size, one should be able to concentratemagnetic particles (e.g., clustered) next to each other and near thedetection side of the microfluidic channel 514 within the field of viewof the objective, 517 which in turn may generate more homogeneous andstrong signals.

In yet another embodiment as shown in FIG. 8C, a pair of magnets 525 maybe placed at a distance of about 0 mm to about 20 mm from a nearestdownstream detection point. The gap between magnets may vary from about0.1 to 10 mm. Magnetic forces generated by this pair of magnets 525 maygradually alter the path of the magnetic particle(s) 521 inside thedroplet 522 as they move toward magnet area in the microfluidic channel524. The magnetic field generated by pair of magnets may force particles521 to position themselves next to each other, preferably in a lineperpendicular to droplet flow and microfluidic channel 524. This designmay be particularly useful for applications where for example an RF-(e.g., TAG) integrated objective is used for signal collection, in whichparticle alignment is in agreement with shape of the light sourcepassing through droplet. By optimizing the flow rate and magneticforces, one should be able to bring magnetic particles to well definedand linear positions to achieve maximum detection efficiency throughobjective 527 per given applications.

In yet another embodiment, as shown in FIG. 8D, two pairs of magnets 534may be placed from two sides of microfluidic channel 537 at a minimumdistance of about 0 mm to about 20 mm from a nearest downstreamdetection point. The gap between magnets may vary from about 0.1 toabout 10 mm. Magnetic forces generated by these two pairs of magnets 534may have more strength and uniformity across the microfluidic channel537,537, thereby leading to more efficient alignment of magneticparticle(s) 531 inside the droplet 533 in a line perpendicular to thedroplet flow in the microfluidic channel 537. This type of alignment maybe useful for applications where, for example, an RF- (e.g., TAG)objective is used for signal collection, in which particles alignment isin agreement with shape of the light source passing through a droplet.By optimizing the flow rate and magnetic forces per defined application,one should be able to bring magnetic particles well defined and linearto get maximum detection efficiency through objective 536 per givenapplications.

In some embodiments, as shown in FIG. 8E, two magnets 545 may be placedon the top and bottom sides of the microfluidic channel 544 at adistance of about 0 mm to about 20 mm from a nearest downstreamdetection point, one at each side of microfluidic channel 544. Magneticforces generated by these magnets 545 may alter the path of magneticparticle(s) 541 inside the droplet 542 as they move toward magnet areain the microfluidic channel 544. By optimizing the flow rate andmagnetic forces per defined application, one should be able to bringmagnetic particles aligned in a linear shape at center of droplet 542well in focus of the objective 547 for applications which requiredetailed analysis of fluorescent signals with minimum destruction frommicrochip or droplet interfaces.

In yet another embodiment as shown in FIG. 8F, two arrays of magnets 555are placed at a distance of about 0 mm to about 20 mm from a nearestdownstream detection point, with one array at each side of microfluidicchannel 554. Magnetic forces generated by these arrays of magnets 555will alter the path of magnetic particles 551 inside a droplet 553 asthey travel toward magnet area in the microfluidic channel 554. Byoptimizing the flow rate and magnetic forces per defined application,one should be able to gradually align the particles in a near-linearshape at the center of the droplet 553 where the particles will be inthe focus of the objective 557 for applications which require detailedanalysis of fluorescent signals with minimum destruction from microchipor droplet interfaces. This design may be preferable when dealing with alarge number of particles or a varying number of high-density particlesinside each droplet 553 in which the use of one magnet on each side maynot provide enough force or uniformity to align particles at the nearestdetection point.

It will be understood by one of ordinary skill in the art that anynumber, size, and/or configuration of magnets (or pairs or magnets orarrays of magnets) may be used as desired based on the teachingsdescribed herein. It should be noted that, in addition to magneticparticle-focusing methods, the concept of focusing intra-dropletparticles can be achieved by using acoustophoresis methods. Theacoustophoresis method for intra-droplet acoustic focusing can be basedon the interaction of sound waves with intra-droplet entities (e.g.,cells, particles) such that the positions of cells or particles inside adroplet can be confined to enable accurate detection by detectors (e.g.,an optical detector). Such acoustophoresis methods may provide severaladvantages over other focusing methods (e.g., conventional hydrodynamicfocusing method), the advantages include (1) the cells or particles mayhe label-free or metal-free, and (2) the acoustic operation conditionsmay be tuned to be gentle, contactless with and minimally harmful tocells.

In many applications with complex biological samples, the purity ofsorted droplets may be critical, i.e., a minimal false positive rateamong sorted droplets may be favored. In some applications, particularlythose involving a long droplet-incubation period and those involving theusage of larger droplets (e.g., with a volume ≥500 pL), some dropletsmay become coalesced between one another (i.e., merged or fused) at astep before the droplets are sorted at a sorting junction, which can becaused by a variety of random or non-random factors such as mechanicalstress, surfactant impurity, and electrical forces. The merged dropletsmay readily break into two or more “daughter” droplets of various sizesright at the bifurcation of a sorting channel, and one of the resultingdaughter droplets may automatically enter the target droplet collectionchannel without any active sorting force, thereby generating falsepositive droplets in the target channel (these false positive events areconsidered “passive sorting”). In applications involving low abundanceevents in a complex sample, these passive sorting events may evenoutnumber the number of true positive. The resulting contamination frompassive sorting may make the post-screening processes very tedious,inefficient, and/or costly. To eliminate or reduce passive sortingevents while maintaining a high sorting rate, a second sorting point canbe added to the same microfluidic chip downstream of a first sortingpoint. In some embodiments, two or more extra-steps of sorting may beused in a serial or tandem manner on the same microfluidic chip.

FIG. 9 shows a system 200 which may comprise an encapsulation unit 201,an incubation unit 202, and two downstream sorting units 203 and 204while coupled in serial. The encapsulation unit, incubation unit, andsorting units may be substantially similar to those described herein. Adispensing unit 223 may optionally be disposed downstream of the secondsorting unit 204, for example in cases where a single cell dispensing isdesired. Similar to what has been described previously for system 100,one or more analytes 206 may be injected into a first inlet of a firstchannel of the microfluidic device 205 and the carrier oil 207 may beinjected from the second inlet with one or more pumps 208 and 209. Cellsand/or particles may be co-encapsulated into droplets 210. Droplets 210may then be collected and incubated in an incubation chamber 211 for apredetermined period of time depending on a specific assay. Afterincubation, droplets 210 may be moved through the microchip for sorting.Optionally, cells/particles may be focused by a permanent or tunableelectric magnet 212 as described herein for enhanced focusing before thefirst point of detection 213, which may use a single-color ormultiple-color laser beams 214 in LIF detection. The detection signalsmay be sent to the data acquisition & processing unit 225 (also referredto herein as a processor) for data analysis. When a droplet isidentified as positive, the data acquisition & processing unit 225 maydeliver a trigger signal to the sorting controller 224 to activate afirst sorting actuator 215 to redirect the droplet to a collectionchannel. For droplets that do not meet the criteria, no trigger signalwill be sent and the sorting actuator 215 will remain off, allowing thefluidic pressure to drive the unwanted droplets into a waste channel216.

The sorted droplets from a first sorting unit 203 are then guided to asecond point of detection 218, which can be based on an optical ornon-optical module. The second sorting actuator 219 can either betriggered by a time-delay based on the droplet traveling time (i.e., thetime for a droplet to migrate from a first to a second point ofdetection), or directly through a sorting controller 224. The finaltarget droplets 222 can be directed to an optional downstream dispensingunit 223 for further analysis.

In another embodiment, the system 230 as shown in FIG. 10 may comprisean encapsulation unit 231, an incubation unit 232, two serial sortingunits 233 and 234, and an optional dispensing unit 253. In theencapsulation unit 231, one or more analytes 237 are injected into oneinlet and the carrier oil 238 is injected from the other inlet withpumps 239 and 240. Cells and/or particles are co-encapsulated intodroplets 241 which are then collected and parked at an incubation Module242 for a period of time determined by a specific assay. Afterincubation, droplets are driven to the first point of detection 244.Optionally, magnetic particles, when provided in the droplets, can befocused by a magnet unit 243 for optimal focusing before the first pointof detection 244. Unlike the detection module setup described in FIG. 9,the first point of detection 244 is configured as a multi-channeldetection module that also serves as a second point of detection at thedownstream of channel direction. The same laser or light source 249 isused for both first and second points of detection, which share a samedetection Module 244. The detection signals are sent to the dataacquisition and processing unit 255 for data analysis. Upon detecting atarget droplet by the detection Module 244, the acquisition andprocessing unit 255 will deliver a trigger signal to the sortingcontroller 254 to activate the first sorting actuator 245, directing thetarget droplet into a collection channel 248. The acquisition andprocessing unit 255 will ignore undesired droplets and the non-targetdroplets will enter a waste channel 247. To perform a serial sorting,the sorted droplets from sorting unit 1 (233) are guided to the secondpoint of detection, which is comprised by the same multi-zone detector244 with a single objective. This setup with a multi-zone singledetector eliminates one extra separate detection point thus makes thedevice more compact. Subsequently, a second sorting actuator 250 can betriggered by a sorting controller 254. The final target droplet 252 canbe directed to an optional downstream dispensing unit 253 for collectionand further analysis. In some embodiments, the emission light in thedetection Module 244 can be split into two downstream detectors (e.g.PMTs). In some embodiments, the emission light in the detection Module244 is detected by a PMT with integrated multi-detection chips (i.e.,multi-channel detector).

For applications involving the screening of millions of droplets, suchas candidate screenings in the content of antibody discovery orsynthetic biology, a screening device with high sample throughput isdemanded to be able to complete the screening process within areasonable time frame, typically on the order of a few hours. However,if the sample is screened by flowing it through a microfluidic channel,throughput, i.e., the flow rate is limited by friction as a laminar flowis preferred. In a brute force approach, the throughput can be increasedby splitting the sample and running it through multiple microfluidicchambers on multiple particle detection/sorting devices in parallel.Yet, this approach is not affordable as operational and consumable costsmultiply with the number of parallel runs. Therefore, it is highlydesired to implement parallel processing in a more streamlined, singlesystem design.

If optical detection is used for sample screening, the field of viewavailable typically exceeds the channel width, e.g., 1 mm field of viewwith a 10×, NA 0.3 objective lens corresponds to a typical channel widthof 0.1 mm. To take advantage of this difference, systems forsimultaneous optical detection in two or multiple microfluidic channelswith a single optical path may be designed as further illustrated herein(e.g., Module 600 shown in FIG. 11 or Module 700 shown in FIG. 16).

With such an approach described in FIG. 11, the assay throughput can bemassively increased (by about 2-20 times). This is highly relevantbecause fast sample screening not only reduces time and cost, but alsoincreases the success of screening applications in part because shorterrun times can reduce the incidence of droplet coalesce and meanwhileimprove intra-droplet cell viability.

For example, in antibody discovery it is common to have only a smallnumber of cells within the entire starting sample to produce an antibodyof interest (e.g., <100 positive cells in millions of starting cells).Therefore, it is favorable to process a large sample size within a shortperiod of time (a few hours or less). To ensure successful isolation ofpositive droplets with reasonable cost and efficiency, the followingcriteria must be met:

-   -   1. High sample flow rate without subjecting the droplets and        cells to unnecessary mechanical stress that could compromise        sample integrity;    -   2. Sample screening within a single device in order to keep cost        and complexity as low as possible.

To meet these requirements on a proposed cell sorting and dispensingsystem such as the one illustrated in FIG. 10, multiple microfluidicchannels can be arranged in parallel on a same chip for simultaneousoptical detection within a single field of view. As shown in FIG. 11,Module 600 enables us to detect optical signals from multiplemicrofluidic channels at the same time. FIG. 16 shows an opticalconfiguration including a beam splitter which similarly enables paralleldetection of optical signals from multiple microfluidic channels at thesame time.

In some embodiments, as exemplified in FIG. 11, the system 230 maycomprise a multi-point detection module 600 configured to detect opticalsignals from two or more channels arranged in parallel (e.g., asdescribed in FIG. 10). The module on the left side of FIG. 11 shows anexemplary single-point detection set up with a single channel (as may beused in any of the systems described herein without parallel detection).In contrast, the module on the right side of FIG. 11 shows a multi-pointdetection set up with two or more parallel channels disposed on/in amicrochip substrate 606 of a microchip 604. The parallel channels may beilluminated by a light source 602 and signals from the channels may bedirected onto a multi-channel detection unit 610 (e.g., a multi-anodephotomultiplier or camera) by a single objective 608.

In some aspects, signals collected from a point of detection in any oneof the disclosed systems may provide informative details for each targetdroplet, such as cell (or particle) number and size, cell viability,spatial distribution of fluorescent intensity, ratios of fluorescentsignals, and other assay readout parameters. While only a small portionof the detailed information is briefly used as real-time sortingcriteria due to time constraint between a detection time point and thefollowing sorting actuation upon a positive event, a significant portionof the collected information are not utilized.

In some embodiments, a comprehensive data analysis can be performed posta sorting and dispensing process without the sorting-associatedtime-constraint. Such a post-processing data analysis may provideadditional information about the sorted droplets to help prioritize thetarget list, for example, by using advanced signal-processing algorithmson the collected data from a point of detection. The ability to furtherprofile or prioritize the sorted and dispensed droplets can be veryuseful for isolating low abundance events. On one hand, one may set“looser” criteria to recover as many targets as possible, which willincrease the false positive rate. On the other hand, one may performpost-processing data analysis and establish additional criteria toeffectively reduce or remove low-quality hits, while retaining thehigh-quality ones. Nonetheless, in order to enable effectivepost-processing data analyses for the target droplets, precise trackingand indexing of individual target droplets during the sorting anddispensing is critical. Such precise tracking and indexing feature canbe achieved or improved by implementing new designs illustrated in thenext few paragraphs.

In some embodiments as exemplified in FIG. 12, the system 260 not onlycomprises a first point of detection 265 and an optional second point ofdetection 271, but also at least one sensor 266 that is used tofacilitate the target droplet tracking and indexing. The at least onesensor 266 will provide precise timing of any passing droplet. Theprecise timing can be effectively synchronized with the timing ofdetecting a target droplet at an upstream point of detection and thetiming of a downstream dispensing of the target droplet. Thissynchronization control can be performed by a data acquisition andprocessing unit 276. In some embodiments, the synchronization control isfurther facilitated by measuring the flow rate of carrier fluid in achannel of a microfluidic chip. In some embodiments, an acceptabledeviation from expected timing is established, such that any unexpecteddroplet that reaches a dispensing point will be simply ignored and notcollected, as long as the droplet arrives at a timing that is beyond anestablished deviation threshold. In some embodiments, a deviationthreshold is based on a statistical model. In some embodiments, adeviation threshold is set as at least one standard deviation of anormal distribution that reflects flow rate fluctuations.

In some embodiments as illustrated in the system 260 (FIG. 12), sortingcriteria (i.e., the thresholds to determine a droplet as a targetdroplet) can be determined and set by a user at the beginning of eachrun based on factors such as signal peak height, area, shape, widthand/or their position in reference to each other within a same droplet.Upon detecting a target droplet at a first point of detection 265, adata acquisition & processing unit 276 will control a sorting actuator268 to redirect the target droplet into a target-collection channel toobtain a sorted droplet. Each target droplet detected at the first pointof detection 265 is tracked and indexed by the data acquisition &processing unit 276, wherein the corresponding processed signal data iscommunicated to and recorded at computer 277. Meanwhile, the sortedtarget droplet will continue its motion to pass through a sensing areaof sensor 266, in which the sensor 266 detects the presence of a passingtarget droplet, such presence or absence information will also beprocessed by the data acquisition & processing unit 276 to provide aprecise timing control to synchronize an upstream sorting step and adownstream dispensing step, as well as any optional point of detectionbefore the eventual dispensing. Following the detection by the sensor,the target droplet will be driven through a microfluidic tubing (e.g., acapillary) 270, where an optional second point of detection 271 can beimplemented with a similar or different laser beam(s) as the first pointof detection; data collected at this step will be also processed by dataacquisition & processing unit 276 and subsequently communicated tocomputer 277. The dispensing Module 272 is triggered in a synchronizedmanner based on the signal data collected from the earlier steps at 265,266 and 271 per user defined settings, which is controlled by dataacquisition & processing unit 276. At the end, a dispensed droplet canbe matched with its corresponding data collected at the first point, andoptionally the second point of detection for the dispensed droplet withthe assistance of computer 277. The collected data can be analyzed toextract useful information for each dispensed droplet in apost-processing manner. This post-processing data-matching capabilityadds significant value to a screening application because it initiates afeedback loop between downstream analysis and screening criteria.

In some embodiments, the at least one sensor is implemented between anupstream point of sorting and a downstream point of dispensing. In someembodiments, the response of a sensor is used as complementary and extradroplet monitoring tool. The sorting and dispense events will mainlyrely on threshold settings used on the first and second points ofdetection, in which it is intended to provide discriminative informationon droplets, with a main focus in the encapsulated cells and/orparticles.

In some embodiments, at least one sensor is positioned at a locationafter a point of sorting but before a point of dispensing along the flowdirection of a microfluidic chip. In some embodiments, the at least onesensor is implemented at a position that is about 3 mm to about 100 mmdownstream of a sorting point, or about 5 mm to about 400 mm before adispensing nozzle of a dispensing point. In some embodiments, the atleast one sensor is integrated into a microfluidic chip. In alternativeembodiments, the at least one sensor is implemented along a microfluidictubing that connects the microfluidic chip to a dispensing nozzle of adispensing module. In further alternative embodiments, at least onesenor is implemented on a microfluidic chip and at least another oneimplemented along a microfluidic tubing that connects the microfluidicchip to a dispensing nozzle of a dispensing module.

In some embodiments, at least one sensor is an optical sensor. In someembodiments, at least one sensor is a non-optical sensor. In someembodiments, both an optical sensor and a non-optical sensor is used.Exemplary non-optical sensors are sensors based on impedance,capacitance, conductivity, microwave, and/or acoustic wave. Examplesoptical sensors include those that are based on transmission orreflection.

In one embodiment, as exemplified in FIG. 13A, two sensors areimplemented in a system 800, wherein at least one of the two saidsensors is a non-optical sensor. The at least one non-optical sensor isimplemented on a microfluidic chip, on a microfluidic tubing (e.g.,capillary), or both (FIG. 13B). In some embodiments, to be used on adevice (e.g., microchip 803; FIG. 13A), a pair of conductive electrodes(828), which are made of, for example, Au, Ag, Cu, Ni or Pt with about 5μm to 150 μm gap and width, are integrated into the microchip substrate(829). In some embodiments, the electrodes can be coated with a thinlayer of microchip material, for example, PDMS, to minimize droplet flowinterruption when the droplet passes a sensor's sensing area. In someembodiments, to be used on a microfluidic tubing (817) that connects amicrofluidic chip to a dispensing nozzle of a dispensing module, a wirecoil unit (830) around a stretch of the microfluidic tubing (e.g.,capillary) is used as a sensor to sense a passing droplet (826) thattravels from an upstream point of sorting to a downstream point ofdispensing. The coil diameter, size, length and loop number of the wirecoil-based sensor can vary. In some embodiments, the wire coil is madeof one or more types of material selected from Ni, Cu, Fe, Ag, and Au.

In another embodiment, as exemplified in FIG. 13B, wherein at least oneof the two said sensors is an optical sensor. The at least one opticalsensor is implemented on a microfluidic chip, on a microfluidic tubing(e.g., capillary), or both (FIG. 13B). In some embodiments (e.g., Module862, FIG. 13B), a beam of light generated by a light source (876) suchas a laser or LED is delivered to the microchip channel through the useof a fiber (879). The beam reflected by a passing droplet will becollected through the same said fiber, 879, passed through abeamsplitter (877), and detected by a sensor component 878, which isconnected with a data acquisition and processing unit (874) forsynchronization control. In some embodiments, to use a sensor on amicrofluidic tubing (867) side in a transmission sensing mode, a lightsource (884) and detection (886) will be positioned at two sides of themicrofluidic tubing where the light beam generated by a laser or LEDwill pass through a lens (885), a moving droplet in the microfluidictubing then a second lens (887) and collected by Module 886, which willbe connected with data acquisition and processing unit (874). To use onthe microfluidic tubing (867) side in a reflection sensing mode, a lightsource (891) and a detection (893) will be positioned at the same sideof the microfluidic tubing where the light beam generated by a laser orLED will pass through a lens (890), a moving droplet in the microfluidictubing then a second lens (892) at about 60 to 120 degree angle, andcollected by 893, which is connected with a data acquisition andprocessing unit (874) for synchronization control.

In some embodiments, similar to the usage described previously for anon-optical sensor, at least one optical sensor is used to provideprecise timing of any passing droplet; the precise timing can beeffectively synchronized with the timing of detecting a droplet at anupstream point of detection and the timing of a downstream dispensing ofa droplet. Such synchronization control can be performed by a dataacquisition and processing unit (e.g., 874 in FIG. 13B). In someembodiments, the synchronization control is further facilitated bymeasuring the flow rate of carrier fluid in a channel of a microfluidicchip.

In general, a droplet sensor is implemented along a flow channel, at thedownstream of a sorting junction and before the nozzle of a dispensingmodule. In some embodiments, at least one sensor is implemented at thedownstream of a sorting junction and before a second point of detection.In some embodiments, at least one sensor is implemented at thedownstream of a second point of detection but before the nozzle of adispensing module. In general, the sequence of implementing at least onesensor and at least one second point of detection along a flow directionof a microfluidic device may be varied or different and shall not beconsidered as limited to the illustrations provided in the Figures.

In some embodiments, any of the systems disclosed herein (e.g., the onesillustrated in FIGS. 1-3, FIGS. 9-10, and FIGS. 12-13B) may comprise anoptional “pico-injector” (or nano-injector) module. The pico-injectormodule may provide injection of a new type of sample and/or reagentsfrom a side channel to a collection channel disposed between an upstreamsorting junction and a downstream point of detection (e.g., between afirst sorting junction and a downstream second point of detection); theside channel is provided with a flow rate ranging from about 0.5% toabout 20% of that for the target droplets 117 in the collection channel.When a target droplet passes through the collection channel to arrive atthe channel segment with a side-opening to the side channel, the newtype of sample and/or assay reagents can be injected from the sidechannel by the pico-injector module; the injected sample and/or reagentsmay become merged with the passing target droplet, wherein the amount ofsample and/or assay reagents and the injection speed may be controlledby the pico-injector module. The sample and/or reagents can beintroduced by the pico-injector to the flow of the target dropletseither in a droplet format or as a direct liquid stream. The sampleand/reagents can be injected by a pressure pump or other pressurecontrollers. The pico-injector can be integrated with a microfluidicdevice (e.g., the microchip 105) after a first sorting junction, orintegrated with a microfluidic tubing (e.g., the microfluidic tubing119) at a position prior to the second point of detection (e.g., thesecond point of detection 121). If a sensor is also used (e.g., in asystem shown in FIGS. 13A-13B), the junction of fluid delivery frompico-injector to a microfluidic channel may be implemented after thesensor's sensing area of the channel to ensure that the pico-injectormainly or exclusively provides fluid delivery (i.e., new sample and/orreagents) to sensed/counted target droplets. The “pico-injected” newsample and/or reagents may react with the existing entities of a targetdroplet, thereby providing new information about the target droplets.The provided new information may facilitate the decision-making at thedispensing module, and/or improve the overall assay efficiency and/oraccuracy.

In yet another embodiment, any of the systems disclosed herein (e.g.,the ones illustrated in FIGS. 1-3, FIGS. 9-10, and FIGS. 12-13B) maycomprise an optional droplet trapping chamber or reservoir to trap, parkor delay the flow of a batch of sorted target droplets post a sortingstep but before a downstream point of detection or before a dispensingnozzle. For example, the sorted droplets 117 shown in FIG. 1 can beparked temporarily in a droplet trapping chamber; the trapping chambercan be integrated as a component of the same microchip 105, at alocation downstream of a collection channel and upstream of the secondpoint of detection 121. If a sensor is also used (e.g., in an exemplarysystem shown in FIGS. 13A-13B), the droplet trapping chamber may bedisposed after the sensor but before a downstream dispensing nozzle. Thetrapping chamber may provide the sorted target droplets 117 a timeperiod to settle and/or pause before the second point of detectionand/or the dispensing nozzle, which in turn may result in more precisedetection at the second point of detection and more accurate dispensingof the target droplets. Parking the target droplets before the secondpoint of detection and/or the dispensing nozzle may also provide usefulkinetic and assay information as extra criteria to select targetdroplets for final dispensing. The shape, geometry and dimension of atrapping chamber may vary; for example, the chamber can be made byenlarging the inner dimension of a collection channel, adding trappingpillars and well arrays on a microfluidic device (e.g., microchip 105),and/or increasing the length of a microfluidic tubing (e.g., using acoil of microfluidic tubing). The droplet trapping chamber canoptionally operate at the conditions described for an incubation unit102 shown in FIG. 1.

In some embodiments, any of the systems described herein (e.g., thesystems illustrated in FIGS. 1-3, FIGS. 9-10, and FIGS. 12-13B) maycomprise both a pico-injector (or nano-injector) module and a droplettrapping chamber. The pico-injector and the droplet trapping chamber maybe disposed in different sequences along the flow direction of amicrofluidic device with respect to one another, but both of them may beimplemented downstream of a sorting junction and upstream of a secondpoint of detection, wherein the said sorting junction is right after afirst point of detection.

Provided is an exemplary process used for implementing the systems,modules, and concepts presented in the current disclosure; thisexemplary process is summarized in FIG. 14.

FIG. 14 illustrates an exemplary process 1000 in accordance with animplementation of the present disclosure. Process 1000 may represent anaspect of implementing the proposed concepts and schemes such as one ormore of the various schemes, concepts and examples described above withrespect to FIGS. 1-13B. More specifically, process 1000 may represent anaspect of the proposed concepts and schemes pertaining to sorting anddispensing single cells for different assay applications using a dropletbased microfluidic system such as, but not limited, to systems 100, 150,190, 200, 230, 260, 800, and 850 while the dispensed droplets will beindexed for precise tracking for downstream analyses.

Process 1000 may include one or more operations, actions, or functionsas illustrated by one or more of blocks 1010 to 1100. Althoughillustrated as discrete blocks, various blocks of process 1000 may bedivided into additional blocks, combined into fewer blocks, oreliminated, depending on the desired implementation. Moreover, theblocks of process 1000 may be executed in the order shown in FIG. 14,or, alternatively in a different order. Furthermore, the blocks ofprocess 1000 may be executed iteratively. Process 1000 may begin atblocks 1010, 1020, and 1030.

At blocks 1010 to 1040, process 1000 may involve introducing an aqueoussolution of analytes for example, cells and/or particles (e.g.,microbeads, nanoparticles) along with the biocompatible oil premixedwith a surfactant, and injecting them into a microfluidic device usingtwo precise pressure- or syringe-pumps. At 1040, a plurality ofwater-in-oil droplets that may include a plurality of cells and/orparticles may be generated under a step called encapsulation or dropletgeneration. The “encapsulated” water-in-oil droplets may be inspectedin-line immediately after the time point of generation for example, byusing a high-speed camera to ensure they meet the requirement of sizeand homogeneity which may vary per assay applications. Failure to meetthe required criteria may result in repeating step 1040 afterre-inspection of system parts, consumables, sample, and reagents, andtroubleshooting. When passed, process 1000 may proceed from block 1040to 1050.

Block 1050 may be considered optional and, depending on the assayapplication, it may or may not be required. This step may be implementedas an extension part of the encapsulation chip, a separate microfluidicchip, or combined with the sorting microfluidic chip. In someembodiments, the 1050 step comprises one or more environmental controlunits, for example a temperature control unit, an oxygen control unit, acarbon dioxide control unit, and/or a humidity control unit. Theincubation step may take several hours. After incubation, droplets areready to be reinjected into a microchip for detection and sorting.

At block 1060, process 1000 may involve a quick system initialization inpreparation for droplet sorting and dispensing. This may be done bycollecting data from various units and modules on the system includingbut not limited, to pumps, lasers, detectors, sensors, data processingunit, controllers, electronics, etc. and may be performed automaticallyor manually. Failure to meet the required performance may resultrepeating the step 1060 after troubleshooting. When passed, process 1000may proceed from 1060 to 1070.

At 1070, process 1000 may involve detection and sorting the selecteddroplets in the sorting unit per user defined settings. This maycomprise detection at first point of detection where the signals will besent to a data acquisition and processing unit for signal processing.Upon detecting a signal reflecting a positive droplet (i.e., “targetdroplet”), the acquisition and processing unit may deliver a triggersignal to a sorting controller which in turn enforces target droplets tomigrate toward the collection channel while others will continue theirmovement per flow direction toward the waste channel.

In some embodiments, a remote focusing lens such as a TAG index lenswith either a regular laser beam or non-diffracting laser beam may beused at this first point of detection to generate sufficient spatialresolution to distinguish small features such as a particle or a cell.In both said embodiments, a beam of light is passed through acylindrical optical element focusing the light into the back aperture ofthe objective lens aimed at the sample, in this case delivered through achannel of a microchip.

In an alternative embodiment, by orienting the illumination/detectionplanes at about 45 degrees (about 60 to about 120 degrees may be used)with respect to the channel direction, we may overcome the limited focaldepth when illuminating/detecting perpendicular to the channel allowingthe of lenses of high NAs while maintaining isotropic detectionefficiency independent of the channel height which in turn enable us todetect lower signals as well as to identify finer spatial features. Inyet another embodiment, one or both objective(s) comprise remotefocusing lenses.

In some embodiments, one, a pair, or array(s) of magnet(s) can be usedbefore the first point of detection in combination with magneticparticles such as beads that are encapsulated together with a cell in adroplet. Magnets can be permanent, tunable electric or a combination ofboth, which can be made of different materials with various shapes,geometries, dimensions and powers. Also, magnetic particles such as butlimited to, beads are widely available while also can be coated withfunctional groups. The magnet(s) may be used in various combinations asexemplified previously in FIGS. 8A-8F and may generate homogeneous andstronger signals when used close (i.e., from about 0 mm to 20+ mm) to apoint of detection.

In yet another aspect, process 1000 may involve directing the sortedtarget droplets from 1070 to an optional second detection and sortingunit (i.e., serial sorting). This optional step 1080 may be critical forapplications involving low abundance events in a complex sample with ahigh probability of getting passive sorting events when only one sortingunit is used (e.g., process 1070). In some embodiments, two or moreextra-steps of sorting can be used in a serial or tandem manner on thesame microfluidic chip. As described previously in FIGS. 9 and 10, step1080 may comprise only one detector, two or more detectors, in whichcase it may also be utilized using a multi-channel detector to coverdetection in both first and second steps of droplet detection in aserial sorting scheme.

The process 1000 may involve step 1090 after target droplets completed asingle (1070) or serial (1080) sorting step. At 1090, the sorted targetdroplets are directed into a microfluidic tubing (e.g., capillary)through an adapter with an outer diameter ranging from about 0.1 toabout 5 mm. The microfluidic tubing can be a capillary made of glass,polymers, or any other materials, pristine or coated, with an innerdiameter preferentially from 0.05 to 0.15 mm. In some embodiments, asecond point of detection is used to verify a sorted target droplet forprecise triggering before dispensing and in some applications to provideadditional information such as spatial fluorescence distribution fromsorted droplets. Similar to the first point of detection, a laser orlight source is used for the second point of detection while signal willbe collected through an objective and detected by an optical detection,for example, a PMT. Similar to what is described above for the firstpoint of detection, a magnet or an array of magnets and RF lenseswith/without non-diffracting beams, either alone or in combination witha prism may also be used as options to enhance detection performance onthis said second point of detection where required by any given assay.Additionally, in some embodiments, a sweeping deflector (as in FIG. 7A)or stroboscopic illumination (as in FIG. 7B) may be used as detectionschemes to eliminate image blurriness and obtain high resolution imagesof targets moving though a microfluidic device at high speeds.

The detection signals from the second point of detection will also besent to the data acquisition & processing for data analysis where adecision may be made on dispensing of target droplets based on analyzingall data received from both first and second points of detection, incomparison with the threshold values set by the operator per assayapplication. The sorting criteria can be based on various factors suchas peak height, area, shape, width and/or their position in reference toeach other within the same droplet based on signal received andcollected via one, two, three, or more laser wavelengths. When adispense requirement is met, the data acquisition and processing unitmay trigger a dispensing unit while all collected data corresponding toeach target droplet will be recorded by the computer for tracking andindexing of that said dispensed target droplet in each vial. Thedispensing unit with a nozzle at the end of the capillary can be anx-y-z moving stage, or a rotating moving stage in which collect targetdroplets in a 96-well plate, 384-well plate, 1536-well plate or anyother multi-well plates, PCR tubes, PCR strips, or any array ofinterest.

In some embodiments, the process 1000 may involve step 1100, the storageof collected target droplets (positive droplets) and delivering them tothe associated labs for downstream analyses while at the same time eachdispensed droplet will be escorted with the corresponding detection dataat all points of detection along with the run log file.

In some embodiments, the process 1000 comprises using at least onesensor that serves to sense or count a moving droplet. At least oneoptical, non-optical, or a combination of both sensors, is implementedbetween an upstream point of sorting and a downstream point ofdispensing to count a droplet passing through the sensor's sensing areain a non-discriminative manner as complimentary and extra dropletmonitoring tool providing precise timing of any passing droplet. Theprecise timing can be effectively synchronized with the timing ofdetecting a droplet at an upstream point of detection and the timing ofa downstream dispensing of a droplet. Such synchronization control canbe performed by a data acquisition and processing unit which can be alsofacilitated by measuring the flow rate of carrier fluid in a channel ofa microfluidic chip.

Although the steps above show a method 1000 of sorting and dispensingdroplets using a microfluidic device in accordance with embodiments, aperson of ordinary skill in the art will recognize many variations basedon the teaching described herein. The steps may be completed in adifferent order. Steps may be added or deleted. Some of the steps maycomprise sub-steps. Many of the steps may be repeated as often asnecessary ensure detection, sorting, and dispensing of target droplets.

Provided are exemplary processes proposed for implementing the systems,modules and concepts presented in the current disclosure, such exemplarymethods are summarized in FIG. 15.

FIG. 15 illustrates various exemplary approaches for implementing thesystems, modules, and processes presented in the current disclosure inFIGS. 1-14. Provided herein is exemplary and FIG. 15 may include moreschemes as shown in A to D. Although illustrated as discrete blocks,various blocks of each schemes of A to D may be extended and dividedinto additional blocks, combined into fewer blocks, or eliminated,depending on the desired implementation. Moreover, the blocks may beexecuted in the order shown in FIG. 15 or, alternatively in a differentorder, and also it may be executed iteratively.

In some embodiments, e.g., as shown in scheme A, the approach maycomprise: generating droplets, incubating droplets, detecting dropletsat a first point of detection on a microfluidic chip using a regularobjective lens, sorting droplets per sorting criteria defined by theoperator, counting droplets by using at least one non-discriminativeoptical or non-optical sensor, detecting droplets at a second point ofdetection on a microfluidic tubing such as a capillary, and finallydispensing droplets using for example an x-y-z dispensing module. Thedispensed droplets in each vial may be delivered to end users fordownstream and off-line analyses while accompanied by the correspondingdata collected at the first and second points of detection, run log, aswell as the supporting information from sensor(s).

In yet another embodiment, e.g., as shown in scheme B, the approach maycomprise: generating droplets, incubating droplets, detecting dropletsat first point of detection on a microfluidic chip using a remotefocusing lens for example a TAG index lens, sorting droplets per sortingcriteria defined by the operator, counting droplets by using at leastone non-discriminate optical or non-optical sensor, detecting dropletsat second point of detection on a microfluidic tubing such as acapillary, and finally dispensing droplets using for example an x-y-zdispensing module. The dispensed droplets in each vial may be deliveredto end users for downstream and off-line analyses while accompanied bythe corresponding data collected at first and second points ofdetection, run log, as well as the supporting information fromsensor(s).

In yet another embodiment, e.g., as shown in scheme C, the approach maycomprise: generating droplets, incubating droplets, detecting dropletsat first point of detection on a microfluidic chip using a prismintegrated with two objectives at about 45 degree at two corners whileone or both can be a remote focusing lens for example a TAG index lens,sorting droplets per sorting criteria defined by the operator, countingdroplets by using at least one non-discriminate optical or non-opticalsensor, detecting droplets at second point of detection on amicrofluidic tubing such as a capillary, and finally dispensing dropletsusing for example an x-y-z dispensing module. The dispensed droplets ineach vial may be delivered to end users for downstream and off-lineanalyses while accompanied by the corresponding data collected at firstand second points of detection, run log, as well as the supportinginformation from sensor(s).

In some embodiments, e.g., as shown in scheme D, the approach comprises:generating droplets, incubating droplets, detecting droplets at firstpoint of detection on a microfluidic chip using a regular objectivelens, sorting droplets per sorting criteria defined by the operator,counting droplets by using at least one non-discriminate optical ornon-optical sensor, detecting droplets at second point of detection viasweeping deflector or stroboscopic illumination on a microfluidic tubingsuch as a capillary, and finally dispensing droplets using for examplean x-y-z dispensing module. The dispensed droplets in each vial may bedelivered to end users for downstream and off-line analyses whileaccompanied by the corresponding data collected at the first and secondpoints of detection, run log, as well as the supporting information fromsensor(s).

FIG. 16 shows a schematic of an optical configuration (Module 700) formulti-point detection where the first and second points of detection areintegrated into a single field of view and the emission light is splitand sent to two downstream detectors, respectively. The opticalconfiguration shown in FIG. 16 may be implemented into system forparallel detection (e.g., system 230 illustrated in FIG. 10), e.g., asan alternative to the multi-point detection design illustrated in FIG.11. The system 230 may comprise a multi-point detection module 600configured to detect optical signals from two or more channels arrangedin parallel (e.g., as described in FIG. 10). The first and second pointsof detection may be integrated/arranged along two parallel oranti-parallel segments of a microfluidic channel(s) under a single fieldof view as shown in FIG. 16. A single cylindrical laser beam (excitationlight) can be provided to illuminate the field of view. Emission lightmay be collected and split by an optical element for detection by twodownstream detectors (PMT 1 and PMT 2) that correspond to the emissionlight at the two points of detection, respectively. For example,emission light may be collected by a single objective, passed through alens, then split by a beam splitter into a first beam and a second beam.The first beam may be directed through a pinhole to a first detector.The second beam may be directed through a pinhole to a second detector.

FIGS. 17A-17B show schematics of systems comprising a section with oneor more bypass channels (i.e., “buffer zones”) to reduce the speed ofmobile droplets for higher resolution imaging by using a camera or acamera-like detector. Any of the systems described herein may comprise abuffer zone to slow the droplets during imaging. For example, the systemcan comprise a widened sorting channel (e.g., buffer zone) to slow thedroplet flow as shown in FIG. 17A. In some embodiments, a serial or anarray of pillars may be provided at the widened channel interface toconstrain the droplets moving along the sorting channel. In someembodiments, the buffer zone may be provided with one or more bypasschannels (e.g., side pores or side channels) downstream of the sortingjunction to reduce the speed of the traveling droplets as shown in FIG.17B. Fluid in the main fluidic channel can partially enter the bypasschannels to effectively reduce the movement speed of droplets, therebyreducing the motion blur during droplet imaging as part of a point ofdetection. In some embodiments, one or two arrays of pillars may beprovided at the interface between the main fluidic channel and thebypass channels to constrain the traveling droplets moving along themain fluidic channel. Droplets with reduced speed in the buffer zone canbe imaged multiple times using a camera or a camera-like detector, aspart of a point of detection. Repetitive short illumination may be usedto further reduce motion blur.

FIG. 18 shows a schematic of an exemplary optical detector with a dualfocusing feature (Module 730), as part of a point of detection. Any ofthe systems described herein may comprise a dual focusing feature. Forhigh-throughput droplet detection and sorting applications,intra-droplet objects (e.g., cells and/or beads) may move within thedroplets passing a detector or sensor and the relative position of theseobjects to the optic focal plane at a point of detection can be random.This may result in poor focusing (i.e., poor signal/noise ratios) andreduced detection efficiency. Dual focusing may provide improveddetection efficiency of intra-droplet moving objects at a point ofdetection. As illustrated in FIG. 18, an optical element can be providedto modulate an excitation laser and achieve dual focusing at a point ofdetection. The optical element may focus on two locations (focus 1 andfocus 2) at two timepoints (timepoint 1 and timepoint 2, respectively)of a traveling droplet inside a fluidic channel on a device. The opticalelement may split a single laser beam into a first beam and a secondbeam and then direct the first and second beams to the point ofdetection to provide dual focusing along the fluidic flow direction.Each droplet can travel through two foci that are closely positioned oneafter another along the droplet flow direction, thereby improving theprobability that at least one focus can yield optical signalsrepresenting intra-droplet objects with improved signal-versus-noiseprofile. The distance between the two foci can be tuned by adjusting thedistance between the objective and the optical device. In someembodiments, the optical element of the beam splitter may comprise afiber optical splitter that can split light into two outgoing laserbeams. In some embodiments, as shown in FIG. 18, the optical element ofthe beam splitter may comprise a birefringent polarizer such as aWollaston prism, which can split light into two linearly polarizedoutgoing laser beams with orthogonal or near orthogonal polarization. Toachieve dual focusing, line-shaped laser illumination for a singlesegment of a microfluidic channel can be provided through splitting thelight with a Wollaston prism. The light may be unpolarized or polarizedat a 45-degree angle before splitting. The distance between theWollaston prism and the objective can be tuned to modify the distancebetween focus-1 and focus-2. Both the side view schematic (FIG. 18A) anda portion of a top view schematic (FIG. 18B) of the optical element areshown.

Additional exemplary embodiments will be further described withreference to the following examples; however, these exemplaryembodiments are not limited to such examples.

EXAMPLES Example 1: Implementation of an Advanced Optical Configurationwith a 45-Degree-Angle Excitation and Emission through a Prism, as Partof a Point of Detection

FIGS. 19A-19D shows an exemplary implementation of an advanced opticalconfiguration as part of a point of detection. The excitation/emissionlight was provided either at an about 45-degree angle relative to amicrofluidic channel's direction, using two objectives positionedperpendicularly to each other at two corners of a prism (FIGS. 19A and19C), or provided at a conventional 90-degree angle (FIGS. 19B and 19D).The implementation of a 45-degree versus a 90-degree angle aredemonstrated in photographs of a section of a microfluidic device withan attached prism on top of the flow channel (FIG. 19A), or a devicewithout the prism (FIG. 19B). Intra-droplet objects were fluorescentcalibration beads of a diameter of about 6 μm and withFITC-fluorophores. The fluorescent signal from the beads was detectedthrough a 535/50 nm band pass filter by using a PMT and was output as avoltage amplitude. Example histogram profiles of signal amplitude ofdetected intra-droplet fluorescent signals are shown in FIGS. 19C and19D, where the 45-degree excitation/emission angle (FIG. 19C) yielded amore homogenous signal size (signal standard deviation ˜0.14 volt), incomparison to a conventional 90-degree angle (FIG. 19D; signal standarddeviation ˜0.37 volt).

Example 2: Implementation of a Point of Detection with Dual Focusing

FIGS. 20A-20B show an exemplary implementation of a dual focusingfeature at a point of detection. Dual focusing-based detection can bedone with a single PMT, for instance, at the first point of detectionbefore a sorting junction. FIG. 20A is an example photograph of a fieldof view, where the focus positions are highlighted with dotted lines andthe relative droplet flow is are shown. The photograph was a snapshottaken based on background scattering light. Line-shaped laserillumination of the same microfluidic channel was generated by splittingthe illumination with a Wollaston prism, as described in FIG. 18. FIG.20B shows an example profile of PMT signals which were detected using acircular 200-μm pinhole (light gray line) and with a 200-μm-wide,1-mm-long slit (dark gray line), respectively. With the long slit,signals for both focus-1 and focus-2 were able to be picked up.

Example 3: Implementation of Multi-Point Detection with Integrated1^(st) Point and 2^(nd) Point of Detection Under a Single Field of View

Shown in FIG. 21A is a snapshot of an optic assembly implemented basedon the multi-point detection schematics shown in FIG. 16. The first andsecond points of detection were integrated/arranged along two parallelor anti-parallel segments of a microfluidic channel(s) under a singlefield of view. A single cylindrical laser beam (excitation light) wasprovided to illuminate the field of view, but the emission light wassplit into two downstream detectors (PMT 1 and PMT 2) that correspond tothe emission light from the two points of detection. FIG. 21B areexample profiles of detected droplets' signals that were detected usingthe foregoing optic assembly.

Example 4: Droplet Imaging at a Point of Detection Inside a Buffer Zone

Using a microdevice with a buffer zone as depicted in FIG. 17B, dropletswere slowed in the buffer zone to obtain improved image resolution. FIG.22 shows example images of a single mobile droplet containing multiplefluorescent objects, at time point 1 (T1), time point 2 (T2), and timepoint 3 (T3), respectively. Images were taken at a point of detection inthe buffer zone using a color-mode (RGB) CMOS camera, each with a 20 ms(millisecond) exposure.

Example 5: Multi-Point Droplet Detection and Indexing

As shown in FIG. 23, droplets were generated by encapsulatingprotein-A-coated microspheres that capture mouse anti-CD3 antibodies(Clone SP34) and fluorescently-labelled secondary antibodies (goatanti-mouse IgGs) with Alexa Fluor-488 (“AF-488”), Alexa Fluor-647(“AF-647”), and Brilliant Violent-421 (“BV421”) dyes. These dropletswere subjected to two points of detection. The first point of detectionwas based on PMT (e.g., upstream of a sorting junction), and the secondpoint of detection was based on a CMOS camera (e.g., downstream of asorting junction). For the second point of detection, the images from 10ms exposure correspond to green (AF-488), far red (AF-647), and blue(BV421) channel for droplet #1, #2, and #3, respectively. Fluorescencewas excited at laser wavelengths 405 nm, 488 nm, and 638 nm,respectively. The individual droplets were indexed as described hereinto keep track of their respective PMT signal (from the first pointdetection) and serial images (from the second point detection). Notethat only one of serial images is shown for each of these indexeddroplets.

In another exemplary assay, the intra-droplet fluorescent objects can bea variety of cells that are labeled with a cell-tracking dye, thatpositively express a fluorescent reporter protein (e.g., GreenFluorescent Protein), or that are fluorescently labelled for a cellsurface marker (e.g., CD4, CD8, CD3, c-Kit and/or HER2).

Example 6: Implementation of Droplet Sensor

FIGS. 24A-24D show an assembly of an exemplary optical sensor and itsimplementation to detect individual droplets, droplet size, dropletspeed, and droplet position along a flow channel. Light of an IR-LED(wavelength at 780 nm) was detected using a Si photodiode in reversebias. The photodiode signal was high pass filtered (f=10 Hz cutoff) andamplified (gain=100). FIG. 24A shows a photograph of an assemblycontaining two optical sensors that was used to detect/sense droplets.FIG. 24B shows a photodiode voltage signal which was generated bydroplets flowing through a glass capillary channel past the Siphotodiode. FIG. 24C shows two sets of photodiode voltage signalsrepresenting droplets, which were detected by using two sensorspositioned at a distance of 35 mm apart along a flow channel. From thesignal time-delay and the known distance between the two sensors, thedroplet velocity was calculated to time the precise dispensing step at adownstream droplet-dispensing module. FIG. 24D shows a histogram ofdroplet size distribution measured by the distance between the positiveand negative spikes shown in FIG. 24C.

Example 7: Screening B Cells that Secrete IgGs Specifically Binding to aHuman Antigen

B cells can be isolated from the spleen or bone marrow of a mouse, orother commonly used animals such as rat and rabbit, after immunizationwith a human antigen (for example, CD3, HER2, IL-17A). Using methodsdescribed in this disclosure including those in FIGS. 14 and 15,immunization-derived mouse B cells, anti-mouse IgG coatedmicroparticles, and Alexa Fluor 647 coated human antigen can beco-encapsulated into droplets with a homogeneous size (e.g., about 60μm). Droplets can be collected in a tube and incubated off chip for 2-3hours.

After incubation, droplets will be reinjected into the sorting unit.Target droplets can be detected based on a far-red peak signal thatreflect the Alexa Fluor 647 fluorescent foci on the microparticles. Thesorting actuator can be triggered by setting thresholds of thefluorescent signals. Then, the sorted droplets can be directed to thesecond point of detection through a microfluidic tubing (e.g.,capillary) for double checking the fluorescent labeled target droplets.After passing the second point of detection threshold, the dispensingunit (x-y-z moving stage) will be triggered to dispense individualdroplets into PCR tubes or strips. The dispensed droplets can be indexedand used for downstream analysis such as single cell PCR and furthervalidation studies.

Example 8: Screening B Cells that Secrete Antigen Specific IgG withMagnetic Particles

In this example, the sample is identical to the one used in Example 1,except that the anti-mouse IgG coated microparticles are magnetic. Thesemagnetic microparticles can be aligned using a permanent or tunableelectric magnet before a point of detection as shown in FIGS. 8A-8E.Compared to the randomly distributed microparticles, the pre-alignedmagnetic microparticles will provide better focusing and thus increasedetection efficiency and accuracy. The sorted droplets will be dispensedand indexed in individual tubes and used for downstream analysis such assingle cell PCR and further validation studies.

Example 9: Functional Assay for Sorting B Cells Secreting Anti-CD3Antibodies to Drive T Cell Activation using Serial Sorting

Primary B cell samples from immunized mouse, microparticles, and Jurkatreporter cells are co-encapsulated into droplets, and incubated forabout 10 hours. Positive B cells secrete anti-CD3 antibodies that canactivate Jurkat reporter cells which consequently produce GreenFluorescent Proteins (GFP) through a transcriptional signaling cascadein the reporter cells as a functional readout. Therefore, targetdroplets can be detected by the presence of green fluorescent signalwhen excited by a 488 nm laser beam, and sorted into the collectionchannel at a point of sorting that can be based on a dielectrophoreticforce or an acoustic force.

The positive hits are usually extremely low for a functional antibodyscreening assay (e.g., <0.01% of starting B cells); therefore, falsepositive rate can be relatively high in part due to droplet-mergingrelated passive sorting. To reduce false positive sorting, a secondsorting unit can be added in a system and method exemplified in FIG. 9to sort droplets again (i.e., serial sorting). The double-sorteddroplets will be dispensed and indexed in individual tubes and used fordownstream analysis such as single cell PCR and identification of thegenetic sequences of resulting antibodies.

Example 10: Functional Assay for Sorting B Cells Secreting Anti-CD3Antibodies to Drive T Cell Activation using Serial Sorting Combined withParallel Detection

In this example, samples are the same as used in Example 3. However, thetwo detection points will share the same objective, which comprises amulti-zone detector such as a multi-zone PMT (e.g., Hamamatsu lineararray multi-anode PMT module H11460-03). This method simplifies theoptical path, leading to a compact design of the sorting device.Downstream dispensing, indexing and analyses can be done after thesecond step of sorting.

Example 11: Screening B Cells that Secrete IgG Specifically Binding toBoth Human BCMA and Monkey BCMA with Signal Indexing for TrackingIndividually Dispensed Droplets

In this example, B cells will be stained with CellTrace Violet whilehuman BCMA antigen conjugated with Alexa Fluor 488 and monkey BCMAantigen conjugated with Alexa Fluor 647 will be used. Microparticlescoated with anti-mouse IgG will be used. All these reagents will beco-encapsulated into droplets together IgG-positive primary B cells thatare enriched from the spleen of a human BCMA immunized mouse.

After about three hours of incubation at 37° C., droplets will bedirected to a first point of detection to detect Alexa Fluor 647 andAlexa Fluor 488 fluorescent foci signal, and then double-positivedroplets will be sorted to obtain a batch of target droplets, and thetarget droplets can be further detected the CellTrace signal that servesto indicate a live B cell and provide a precise timing of a targetdroplet to synchronize with its detection at an upstream point ofdetection. Only a target droplet with a live B cell will be dispensedinto a multi-position collector, such as a 96-well PCR plate. Bysynchronizing each target droplet from its upstream first point ofdetection and a downstream second point of detection, the collectedfluorescent signals (i.e., blue, green and red) of each said targetdroplet can be perfectly correlated to enable a very informativeanalysis of a single B cell within any target droplet.

Example 12: Implementation of Remote Focusing at a Point of Detection

The objective lens of one of disclosed cell sorting and dispensingsystems in this disclosure while interfacing with the sample will beilluminated with a parallel beam through a beamsplitter followed by acylindrical lens (for example, Thorlabs N-BK7 Mounted Plano-Convex RoundCyl. Lens, cat #LJ1629RM-A). The illumination beam will be selected of adiameter large enough to fill the back aperture of the objective lens aswell as the aperture of the TAG lens (e.g., TAG Optics TAG Lens 2.0Optimized for Visible Spectrum). Along the non-focusing axis of thecylindrical lens, the beam will be focused into a tight spot defined bythe NA of the objective lens (e.g., Olympus 10× NA 0.3 WD 10 mm airimmersion lens). By changing the focal length of the TAG lens, the focalplane position at the sample can be rapidly translated and the sameresolution can be realized along the entire axial extension of thechannel.

Light from the sample will be captured with the same lens, separatedfrom the illumination light via the beam splitter and focused into anaperture with a size corresponding to the lateral extension of the focalspot times the effective magnification of the objective lens beforebeing detected with a PMT (e.g., Hamamatsu photosensor module #H10721).

This confocal detection scheme should efficiently suppress out-of-focussignal. Along the focusing axis of the cylindrical lens, the parallelillumination beam will be focused into the back focal plane of theobjective lens resulting in a demagnification of the beam matching thewidth of the channel after exiting the objective lens. Along thisdirection, the aperture before the detector will be set to be largeenough to cover the magnified image of the channel. The TAG lens will belocated in the back focal plane of the objective lens coinciding withthe focus position of the cylinder lens where the beam diameter shouldat its thinnest. Therefore, the effective aperture of the TAG lens willbe very low and the beam propagation should be only minimally affectedby TAG lens refocusing along the focusing axis of the cylinder lens.

Example 13: Suggested Implementation of Two-Sided Illumination/Detectionat a Point of Detection

In this example, our cell sorting and dispense system consists of twoobjective lenses (OL), OL1 and OL2 while interfacing with the sample viaa prism (for example, Thorlabs Dove Prism, 15 mm, N-BK7, ARC: 350-700 nmPS992-A). Both objective lenses will be illuminated with a parallel beamthrough a beamsplitter followed by a cylinder lens. Along thenon-focusing axis of the cylinder lens, the beam will be focused into asheet defined by the numerical aperture of the objective lens as well asthe illumination beam diameter. Light from the illuminated plane willthen be captured with the opposing lens and separated from theillumination light via the beamsplitter. The signal will then be focusedonto a PMT.

Example 14: Implementation of Multi-Zone Detection Modules at a Point ofDetection

In this example, our cell sorting and dispensing system consists of aconstant or pulsed illumination source such as a continuous-wave (CW)laser or q-switched laser (e.g., Crystal Laser Diode PumpedUltra-compact Q-switched Lasers). The targets will be imaged in amicrofluidic device with objective lens and a tube lens on to anelectron-multiplying CCDs (EMCCD) or scientific CMOS (sCMOS) camera chip(e.g., Photometrics Prime 95B back-illuminated sCMOS camera).

At minimum, two ways can be used to avoid motion blurriness. A sweepingdeflector (e.g., Cambridge Galvanometer Scanner cat #62xx-H and 82xxK)in the detection path can be used to compensate for the particlemovement and keep its image position constant on the camera chip duringthe camera exposure cycle. Alternatively, a brief illumination pulse canbe used to limit signal photon generation at the sample plane to a timespan in which the target has not moved significantly compared to thespatial resolution demanded.

Example 15: Alternative Implementation of Multi-Zone Detection Modulesat Point(s) of Detection

When viewed from the side, the design of our system and its detectionmodules is similar to a conventional optical detection within a singlemicrofluidic channel. A parallel beam will pass through a cylinderoptical element, viewed along the non-focusing axis. After passing abeam splitter, the beam will be focused into the microfluidic channelvia an objective lens. Hence, a thin sheet of light will illuminate thechannel perpendicular to the direction of flow.

Signals such as fluorescence will be picked up from the sample passingthe channel at the location of the light sheet by the same objectivelens, are reflected off the beam splitter and by passing through anaperture that blocks signals from out-of-focus planes. After passing theaperture, the signal will then be re-focused onto the active area of asuitable detector, such as a PMT.

When viewed from the front, i.e., looking at the cross-section of thechannel(s), a single channel can be typically illuminated along itsentire width. However, that width will be usually much smaller than thefield of view of the optical arrangement. To utilize the full field ofview to increase sample throughput, the sample can be split up withinthe microfluidic chip into multiple channels located next to each other(e.g., three channels of 0.1 mm width at a spacing of 0.1 mm resultingin a total demanded field of view of 0.5 mm).

Along this direction, the illumination beam will also be split intomultiple parallel beams, for example, with a mask or a micro-lens array.Each beam will be focused into the back focal plane of the objectivelens by the cylindrical optical element. The spacing and widths of thebeams should be configured such that each channel is illuminated alongits width.

Alternatively, a single beam can be used to illuminate all channelssimultaneously. The advantage of using multiple beams is that the gapbetween channels will not be illuminated resulting in reducedbackground. Signals from all channels will be picked up by the objectivelens and reflected off the beam splitter. With the tube lens, the lightfrom each channel will be refocused into a bundle of parallel beams. Anaperture can be used to spatially filter the bundle of beams to reducebackground and crosstalk between channels. With another lens, eachbundle will then be focused on a separate active area of a multi-zonedetector such as a linear PMT array. Optical filters can be added to thedetection path to select for specific wavelength bands. As analternative to a multi-zone PMT, a camera could be used as detectorwhere the light from the different fluidic channels is focused ondifferent regions of the camera chip.

Example 16: Isolation of Antigen-Specific T Cells Based on CytokineSecretion Profile

Secretion of cytokines are a functional marker of many immune cells. Forinstance, CD8-positive cytotoxic T cells are known to become activatedand secrete signatory cytokines such as IL-2, IFNγ, and TNFα, upon the TCell Receptor (TCR) interaction with a cognate peptide-MHC complexpresented by an antigen presenting cell or a cancerous cell where thepeptide is processed from a tumor neoantigen such as MART-1 andNY-ESO-1. Typically, antigen-specific T cells are rare events (<0.01% ofall T cells). To screen for a rare NY-ESO-1-specific cytotoxic T cellthat may be present in a human blood sample or a patient-derived tumorinfiltrating lymphocyte (TIL) sample, a NY-ESO-1 peptide (e.g., thepeptide sequence comprising SLLMWITQV) can be presented by a MHCmolecule (e.g. a HLA-A2 variant) on a model cell such as K562, to obtainan engineered antigen presenting cell, K562^(NY-ESO-MHC). Then,K562^(NY-ESO-MHC) cells can be co-encapsulated with a pool of human Tcells derived from a donor or patient blood sample, as well as IFNγcapture microbeads (coated with a first anti-IFNγ antibody) and a secondanti-IFNγ antibody labeled with Alexa Fluor 488-conjugated, such thatthe vast majority of droplets will each receive precisely zero or onecandidate T cell, one or two K562^(NY-ESO-MHC) cells, and at least oneIFNγ capture microbead. Upon cognate interaction of a T cell and theencapsulated K562^(NY-ESO-MHC) cell, the T cell may become activatedwhich can be detected based on positive fluorescent focus formation onan IFNγ capture microbead in the droplets. Then, the focus-positivedroplets, which are of low abundance, can be detected, sorted, anddispensed by using one or more exemplary systems described herein. Thedetected fluorescence signal data can be further processed and matchedwith each individual dispensed droplet to facilitate the analysis andidentification of single T cells inside the recovered droplets.

Example 17: Identification of Circulating Tumor Cells (CTCs)

Circulating tumor cells (CTCs) represent an important topic for tumorliquid biopsy field. Circulating tumor cells (CTCs) are cells that haveshed into the vasculature or lymphatics from a primary tumor and arecarried around the body in the circulation. CTCs thus constitute seedsfor the subsequent growth of additional tumors (metastases) in vitaldistant organs, triggering a mechanism that is responsible for the vastmajority of cancer-related deaths. CTCs may serve as an important markeror diagnostic target for early tumor detection, tumor diagnosis, patientstratification, treatment monitoring, disease progression monitoring,and prognosis. Blood samples may be encapsulated in droplets withdetection reagents for CTCs and subjected to immuno-detection or PCRdetection to identify the ones with positive signals using the disclosedmethods and systems in this disclosure. Identified (target) droplets)can then be sorted for further analysis as described herein.

Example 18. In Vitro Screening of Target Specific Compounds

Any of the disclosed exemplary systems and methods described herein canbe used for the selection and screening of a target specific compound. Acompound library comprising a plurality of compound-loaded beads thatare also barcoded (i.e., the so-called “one kind of compounds on onebead”, or, “one-compound-one-bead”) can be individuallycompartmentalized with a target-expressing reporter cell within aplurality of droplets. If the provided target is bound by a specificcompound in a same droplet, the reporter cell will produce a signalwhich can be a fluorescent molecule. The droplets can be detected andsorted using any of the disclosed exemplary systems and methods in thisdisclosure, to precisely identify the barcode on the recovered dropletsand subsequently identify the candidate compound(s).

Example 19: Screening of Genome-Edited Cells

Any of the disclosed exemplary systems and methods described herein canbe used for the screening of single cells (e.g., T cell, B cell,dendritic cells, natural killer cells, stem cell, beta cell, neuroncells, yeast, bacterium, etc.) that have been subjected to a CRISPR-cas9mediated genome editing. Edited (i.e., engineered) cells can be provideda readout assay to indicate a successful editing event. Individualedited cells can be encapsulated with assay reagents (if any) intoindividual sub-nanoliter droplets which may then be introduced into amicrofluidic device for subsequent measurement of the readout signalthat reflect an edited cell's function or phenotypes. Exemplary readoutsignal can be a GFP-reporter, a luminescent reporter, a fluorogenicsubstrate, and/or a fluorescent labelled detection bead. Target dropletswith user-established readout criteria can be identified and recoveredusing any of the disclosed systems and methods described herein.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value, unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that throughout the application, datais provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-20. (canceled)
 21. A system for processing droplets, the systemcomprising: (a) a microfluidic device comprising a first channelconnected to a second channel by a first sorting junction; (b) one ormore droplets, wherein a droplet of the one or more droplets comprisesat least one cell, at least one particle, or both; (c) a first sensorcorresponding to a first point of detection disposed along the firstchannel configured to detect one or more signals generated from thedroplet of the one or more droplets; (d) a second sensor correspondingto a second point of detection disposed along the second channel,wherein the second sensor is configured to detect one or more signalsgenerated from the droplet of the one or more droplets; and (e) aprocessor configured to index the droplet of the one or more dropletswith the one or more signals of the first and the second sensor at thefirst and second points of detection.
 22. The system of claim 21,further comprising one or more lasers or laser-like light sources togenerate illumination at the first point of detection.
 23. The system ofclaim 21, further comprising an optical element configured to providedual focusing along the first channel at the first point of detection.24. The system of claim 23, wherein the optical element comprises anoptical fiber splitter or a birefringent polarizer configured to splitan energy beam generated by the one or more lasers or laser-like sourcesinto a first beam and a second beam and direct the first and secondbeams to the first point of detection
 25. The system of claim 21,wherein the first sensor comprises a fast-response optical detector. 26.The system of claim 25, wherein the fast-response optical detectorcomprises a photo multiplier tube (PMT) or an avalanche photodiodedetector (APD).
 27. The system of claim 21, wherein the second sensorcomprises a camera or a camera-like detector.
 28. The system of claim21, wherein the second sensor is configured to generate two or moreimages of the droplet of the one or more droplets, wherein the two orimages comprise a signal generated by stroboscopic illumination.
 29. Thesystem of claim 21, wherein the processor is configured to synchronize adispensing nozzle with one or more of the first signal and the secondsignal.
 30. The system of claim 21, further comprising an opticalassembly configured to provide stroboscopic illumination at the secondpoint of detection.
 31. The system of claim 30, further comprising anupstream sensor corresponding to a third point of detection disposedalong the second channel between the sorting junction and the secondpoint of detection, wherein the upstream sensor is configured to providea precise timing trigger to the optical assembly to trigger thestroboscopic illumination.
 32. The system of claim 30, wherein the firstsensor is configured to provide a precise timing trigger to the opticalassembly to trigger the stroboscopic illumination.
 33. The system ofclaim 21, wherein the microfluidic device further comprises a wastechannel in fluid communication with the first channel.
 34. The system ofclaim 21, wherein the one or more droplets comprise water-in-oildroplets.
 35. The system of claim 21, wherein (e) is performed in asorting module disposed downstream from the second point of detection.36. The system of claim 30, wherein the stroboscopic illuminationprovides a short illumination within a range of about 0.5 to about 50milliseconds.
 37. The system of claim 21, wherein the one or moredroplets comprise a droplet volume from about 0.01 nanoliters (nL) toabout 10 nL.
 38. The system of claim 21, wherein the first channelcomprises a size of at least 35 micrometers.
 39. The system of claim 27,wherein the camera or camera-like detector comprise a charge coupleddevice (CCD), photodiodes, complementary metal-oxide semiconductor(CMOS) cameras, or any combination thereof.
 40. The system of claim 32,wherein the stroboscopic illumination is generated by modulating acontinuous-wave (CW) laser.